CERTAIN ASPECTSOF TYROSINE METABOLISMIN THEYOUNG. I. THE DEVELOPMENTOF THE TYROSINE

OXIDIZING SYSTEMIN HUMANLIVER'

BY NORMANKRETCHMER,S. Z. LEVINE, HELEN McNAMARA,ANDHENRYL. BARNETT

(From the Department of Pediatrics, The New York Hospital-Cornell Medical Center,New York, N. Y.)

(Submitted for publication September 12, 1955; accepted October 31, 1955)

The major pathway for tyrosine catabolism inthe animal is the non-oxidative transamination oftyrosine to p-hydroxyphenylpyruvate and its sub-sequent oxidation to homogentisate. The ho-mogentisate is oxidized to fumarylacetoacetate(maleylacetoacetate), which is finally hydrolyzedto fumarate (malate) and acetoacetate. The en-zyme system responsible for this catabolism, ten-tatively called the tyrosine oxidizing system, issoluble and a partial purification can be accom-plished with high speed centrifugation of homoge-nates of liver (1) and kidney (2). When thesepreparations are used it has been observed (3)that one mole of tyrosine will be completely oxi-dized to one mole of acetoacetate and one moleof fumarate, with the utilization of two moles ofoxygen. In order to obtain maximal tyrosinetransaminase activity it is necessary to add a-keto-glutarate and pyridoxal phosphate in vitro. Theoxidation of the formed p-hydroxyphenylpyruvaterequires ascorbic acid (1, 4, 5) as a cofactor or anequivalent oxidation-reduction substitute such asdichlorophenolindophenol (6, 7).

Clinically, it was shown (8, 9) that prematureinfants maintained on a diet high in protein with-out added ascorbic acid excreted p-hydroxy-phenylpyruvic acid in their urine. Similarly,p-hydroxyphenylpyruvic acid was excreted in theurine of scorbutic adults (10, 11). The additionof ascorbic acid to the diet of both groups resultedin the disappearance of this substance from theurine. It was also observed that when largedoses of pteroylglutamic acid were given to scorbu-tic guinea pigs (12), monkeys (13), and prema-

1 This investigation was supported in part by researchgrants from The National Institute of Arthritis andMetabolic Diseases of the National Institutes of Health,Public Health Service (A-389), from The New YorkHeart Association and from The Jaffe Fund for Pre-mature Infants.

ture infants (14) there was a decrease in ketoacid excretion. ACTH, after a latent period, alsodecreased the excretion of p-hydroxyphenylpy-ruvic acid by premature infants (15) and scorbu-tic guinea pigs (16). The mode of action ofpteroylglutamic acid and ACTH is still obscure.In contradistinction to the above findings it wasobserved that in two of the three reported adultcases of tyrosinosis there was no effect of ascorbicacid on the urinary excretion of tyrosine or ofp-hydroxyphenylpyruvic acid (3).

These investigations are concerned with the re-lationship of growth of the organism to the de-velopment and action of the tyrosine oxidizingsystem. The data obtained from premature in-fants, immature rats, and older subjects will beused in an effort to explain the previous results interms of a developing enzyme system.

METHODS

1. SubjectsSamples of liver from premature infants, newborn in-

fants and adults were obtained at autopsy.2 All donorswere clinically free of hepatic disease and their liversshowed no gross evidence of liver pathology. Themothers of all premature infants had uneventful preg-nancies. Special precautions were taken to obtain thelivers from the premature infants as soon after deathas possible. These infants were removed to the morguerefrigerator; the autopsy was promptly performed andliver samples obtained.

Livers from all subjects were immediately placed in achilled beaker which was kept on ice and transportedto the laboratory for immediate preparation. The dataon human subjects are summarized in Table I.

Samples of adult animal livers were obtained fromthe Long-Evans strain of rats following sacrifice by ablow to the head. The liver was quickly removed andrinsed with ice-cold 0.9 per cent KCI to remove adherent

2 The authors acknowledge the assistance of Dr. Ar-thur Carlson of the Department of Pathology, and thePediatric House Staff in obtaining the specimens.

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DEVELOPMENTOF THE TYROSINE OXIDIZING SYSTEM IN LIVER

TABLE I

Summary of clinical data

AutopsyEstimated hoursgestational Wt. S.A. after

Subject Sex Age age-days kg. M' Cause of death death

I. Premature infantsP-10 M 4hrs.P-12 M 51 hrs.P-14 M 168 hrs.Pj15t* M 24 hrs.

P-21 F 55 hrs.

P-23t M 7 hrs.P-24t M 28 hrs.

P-31 F 6 hrs.

P-32 F 7* hrs.

P-34 F 16 hrs.

P-1 M 10 hrs.

178 0.89233 1.94229 1.48

1.40

176 0.87

193 1.01193 1.27

228 1.13

172 0.72

189 0.75

169 0.72

II. Full-term infantsNB-19 M 36 hrs.

NB-35 M 20 hrs.

III. ChildC-23

IV. Adults

F 11 yrs.

A-13 M 55 yrs.A-13 F 70 yrs.

A-22 F 65 yrs.

A-25 M 37 yrs.

A-29 M 58 yrs.

.084

.151

.124

.119

.083

.093

.110

.101

.072

.074

.072

Immaturity, atelectasisImmaturity, atelectasisImmaturity, atelectasisImmaturity, atelectasis, hyaline

membrane diseaseImmaturity, atelectasis, multiple

hemorrhagesImmaturity, atelectasisImmaturity, multiple hemor-

rhages, atelectasis, emphy-sema

Immaturity, atelectasis, hyalinemembrane disease

Immaturity, atelectasis, subduralhemorrhages

Immaturity, kernicterus, atelec-tasis, ulcerations of ileum

Immaturity, atelectasis, hyalinemembrane disease

2.76 .198 Atelectasis with multiple visceralhemorrhages

3.22 .221 Aspiration pneumonia, multiplehemorrhages

28.4 .892 Multiple congenital anomalies,hydronephrosis, agenesis of leftkidney, hypertensive cardio-vascular disease

64.0111

39.0

56.0

56.0

1.821.56

1.46

1.77

1.70

Astrocytoma-resectedDiffuse hemorrhagic encephalop-

athy, cardiovascular diseaseCoronary arterial occlusion with

myocardial infarctionHypertensive cardiovascular dis-

easeFibro-caseous pulmonary tuber-

culosis, cor pulmonale with ar-teriosclerotic heart disease

1i1*73

23

3*7

3

3}

6

2*

3*8

9*

1414

12

5

10*

* t = twin.

blood prior to grinding. Infant and newborn rats weresacrificed by decapitation or decerebration and ex-sanguinated. Fetal rats were removed from a pregnantanimal and decapitated. The livers from all of thegroups were removed immediately for tissue prepara-tion except in the case of those rats which were per-mitted to remain for varying periods in the cold room(temperature 2 to 4°C) or at room temperature (26 to28°C) to assay the effect of these factors on the hepatictyrosine oxidizing system. Only litter mates were com-pared in these experiments.8

8 The effect of varying conditions following autopsy onthe tyrosine oxidizing system Was studied by killing in-fant rats by different techniques and exposing them afterdeath to room temperature for varying time intervals

2. Preparation of tissueHuman and rat livers were immediately placed in a

dish on ice in the cold room for prompt and completepreparation of samples. Generally, the human liver waspressed through a hand-tissue press with a sieve of 1 mm.diameter holes to remove connective tissue mechanically.(17). No differences in oxidative activity resulted whenthe animals were sacrificed with and without completeexsanguination. Rats exposed to room temperature forthree hours showed no change in oxidative activity, butexposure for six hours resulted in a decrease in activity.The decrease was proportional to a rising level ofhomogentisic acid which was undoubtedly due to thedecrease in the pH of the liver and the subsequent in-hibition of homogentisic acid oxidase (3).

237

N. KRETCHMER,S. Z. LEVINE, H. MCNAMARA,AND H. L. BARNETT

The resultant mash was ground for two minutes in aWaring Blendor with one to three volumes of ice-cold0.9 per cent KCI. No difference in oxidative activitywas observed whether the homogenates were preparedin the Waring Blendor or the Teflon-glass homogenizer.The rat tissue was homogenized in the Teflon homog-enizer because of the small amount of sample. In thecase of the infant rats, it was necessary to pool two tofour livers to provide a sufficient quantity of tissue. Fol-lowing homogenization the volume was measured andaliquots were centrifuged in the Sorvall SS-1 centri-fuge at 18,000 X g for 15 minutes. The resultant super-natant fluid or soluble fraction was collected and the vol-ume measured. It was found that a force of 100,000 X gfor 30 minutes in the Spinco centrifuge Model L did notdecrease the oxidative activity, indicating that the en-zyme was soluble (1). In some instances the solublefraction was dialyzed against tap water in the cold over-night and the activity of the dialyzed material was meas-ured. This modification of the procedure suggested byKnox and LeMay-Knox (1) was found to be simpler andless erratic than the use of acetone powders (18) orsucrose as a solvent (19).

3. Measurement of oxidative activityMeasurements were made with the total homogenate,

the soluble fraction resulting from high-speed centrifuga-tion and the dialyzed soluble fraction. Samples (0.30 to1.00 ml.) were placed in previously prepared Warburgflasks with sidearms, the flask contents being essentiallythose described by Knox and LeMay-Knox (1). Be-sides the liver samples, the flasks contained 2.0 ml. of0.2 M phosphate buffer pH 7.4, 8 1AM of tyrosine, and0.2 ml. of an ascorbic acid solution (.250 to 5 mg.) ad-justed to pH 7.4. The sidearm contained 0.3 ml. ofa-ketoglutarate (30 uM) adjusted to pH 7.4 and 0.2 ml.of pyridoxal phosphate (60 lg.).4 The center well con-tained 0.2 ml. of 20 per cent KOHon a strip of filterpaper in order to absorb carbon dioxide. Glass distilledwater was added to the main central compartment tobring the total volume to 3.7 ml. A control flask lack-ing tyrosine but containing all of the other reactantswas run with each tissue sample. The "endogenous"oxygen uptake was measured and deducted from the oxy-gen uptake of the flasks containing tyrosine. Measure-ments were made at 37.3°C and were started after anequilibration period of 10 minutes at which time the con-tents of the sidearms were tipped into the main portionand the stopcocks were closed. Readings were takenat 10-minute intervals for a total period of 60 minutes.When measurements were made in an atmosphere ofnitrogen the flasks were "gassed" on the bath during the10-minute equilibration period. Following closure ofthe stopcocks, readings were taken to preclude the ex-traneous uptake of oxygen.

4 The authors wish to thank Dr. Kenneth Tsuboi forhis generous contribution of pyridoxal phosphate.

4. Chemical determinations

Nitrogen determinations were made on aliquots ofthe original liver tissue by the Ma-Zuazaga modificationof the micro-Kjeldahl method (20). An additional ali-quot of the total homogenate was treated according to amodification of the Schneider trichloroacetic acid frac-tionation method (21) and desoxyribonucleic acid was de-termined on the fraction remaining after the sample wasfirst treated with an equal volume of ice-cold 10 per centtrichloroacetic acid and alcohol-ether. The final pre-cipitate was heated with 5 per cent trichloroacetic acidfor 15 minutes at 90°C and the desoxyribonucleic acidwas determined in the supernatant fluid with the di-phenylamine reaction (22).

Immediately following completion of measurementsof oxidative activity, the contents of the Warburg flaskswere removed and aliquots were placed in equal vol-umes of trichloroacetic acid (TCA) and metaphos-phoric acid to make 5 per cent solutions. The pro-tein precipitate was removed from both of these sus-pensions by centrifugation. From the supernatant fluidof the TCA precipitation, an aliquot was taken for thedetermination of Briggs reacting material (p-hydroxy-phenylpyruvic acid, homogentisic acid, and also ascorbicacid) according to a modification of the method of Medes(23). In order to remove interference from ascorbicacid an aliquot from the trichloroacetic acid solutionwas extracted three times with peroxide-free ether. Thisether extract containing p-hydroxyphenylpyruvic acidand homogentisic acid was evaporated to dryness andthe Briggs' reaction was performed as outlined byMedes. Similar treatment of pure solutions of p-hydroxy-phenylpyruvic acid gave recoveries of approximately 70to 75 per cent. The original trichloroacetic acid filtrateprior to ether extraction was utilized directly for thedetermination of homogentisic acid by the iodometricmethod of Neuberger and Webster (24).

In order to determine micro-quantities of homogentisicacid, .001 N thiosulfate and .001 N iodine were used. Toestimate maleylacetoacetic acid, another aliquot of thetrichloroacetic acid extract was read in the BeckmanDU spectrophotometer with a blank prepared from theTCA extract of the specific control flask. Acid solutionsdepress the ultraviolet absorption of maleylacetoaceticacid (3) but readings of samples showing oxidative ac-tivity at wave lengths of 300 my to 345 mn showed aband which was not present in the tissue blanks or insamples incubated in an atmosphere of nitrogen. The ab-sorption of the p-hydroxyphenylpyruvic acid was com-pensated for by the addition of an equivalent amount ofp-hydroxyphenylpyruvic acid to the tissue blank againstwhich the samples incubated in nitrogen were read.Under these conditions the presence of an absorptioncurve at 300 mny to 345 my. was taken as an estimate ofthe presence of maleylacetoacetic acid.

The metaphosphoric acid extract was used for meas-urement of p-hydroxyphenyl compounds (24): tyrosineand p-hydroxyphenylpyruvic acid. To avoid turbidity,these samples were read as soon as they became optically

238

DEVELOPMENTOF THE TYROSINE OXIDIZING SYSTEM IN LIVER

constant, approximately three minutes after the sodiumnitrite was added. The remainder of the metaphosphoricacid extract was used for the formation and determina-tion of 2-4 dinitrophenylhydrazones according to themethods of Cavallini and Frontali (25). The meta-phosphoric acid filtrate was first extracted three timeswith peroxide-free ether in order to remove p-hydroxy-phenylpyruvic acid and leave the a-ketoglutaric acid inthe residue. It was found that a-ketoglutaric acid, be-ing in much greater concentration than the aromaticketo acid, interfered with the chromatograms in the ab-sence of ether extraction. The hydrazones which wereformed on addition of 2-4 dinitrophenylhydrazine werechromatographed with n-butanol (25) as the movingsolvent on Whatman No. 3 strips. A few drops offormic acid were placed in the drum to prevent tailing.The p-hydroxyphenylpyruvic acid migrated as two spotsdue to the syn-anti configuration and gave Rf values of0.83 and 0.96. Pure homogentisic acid and p-hydroxy-phenylpyruvic acid for use as standards were obtainedthrough the courtesy of Dr. W. Eugene Knox and thelatter was also purchased from the Homberg Chemie-werk, Frankfurt am Main.

RESULTS

The data in Table I show that 10 of the 11 liverspecimens from premature infants were obtained

within 7 hours after death. The weight of the in-fants ranged from .72 kg. to 1.94 kg. and theirgestational age, from 169 to 233 days with a meanof 196 days. Specimens from full-term infantsand adults were obtained within 14 hours afterdeath.

The data in Table II present the oxidative ac-tivities obtained with the total homogenate, thesoluble fraction derived from high speed centri-fugation and the dialyzed soluble fraction. Sincethis enzyme system is soluble it will be notedthat when enzyme activity is rated on the basis ofnitrogen it consistently increased in the adult andfull-term infant livers as the nonactive particulatematter was removed. In contrast, this increasewas noted in the soluble fraction of only 2 of the 11premature infant livers and in terms of thenitrogen standard was negligible. The liver fromthe premature infant obtained 23 hours afterdeath (P-21) showed virtually the highest activityboth on the basis of wet weight and nitrogen.The hepatic activity of the full-term infants andthe adults was 3 to 20 times greater than that of

ILE II

The activity of the tyrosine oxidizing system in fractions of human liver

DialyzedSoluble soluble

Homogenate Homogenate fraction fractionSubject Al. 02/30'/gm. wet wt. ;J. 02/30'/mg. NI. Premature infants

P-10 0 0 0 0P-12 6 0 2 2P-14 59 2 4 4P-iS 0 0 0 -P-21 81 4 4P-23 51 2 1 2P-24 9 0 0P-31 - 1P-32 18 1 1 1P-34 0 0 0 -P-41 - 0

Average 25 1 1 1

II. Full-term infantsNB-19 51 2 5 -NB-35 63 3 5 5

Average 57 3 5 5III. Adults

C-23 240 9 26 17A-13 732 26 36 27B-13 192 8 21A-22 164 7 11A-25 204 7 13 25A-29 316 10 28 57

Average 308 12 23 32

239

N. KRETCHMER,S. Z. LEVINE, H. MCNAMARA,AND H. L. BARNETT

TABLE III

Activity of the tyrosine oxidizing system of the soluble fraction with various standards of reference

Total liver activityOxygen uptake gd. 02/30'/M2 body

S.A. X10'-Number Number Number

Of of of Numbersubjects Range Average subjects Range Average subjects Range Average of

4./30'/gm. wet wt. ;d./30'/mg. N 1d./30'/mg. DNA* subjects Range Average

Prematureinfants 10 0-80 21 11 0-4 1 2 6-15t 11 10 0-45 15Full-term infants 2 73-79 76 2 5-5 5 2 22-34 28 2 44-51 48Adults 6 140-612 343 6 11-36 23 1 135t 6 152-483 318

* The DNAwas determined on the total homogenate; as expected, no DNAwas found in the soluble fraction.t The values for ul. 02/30'/mg. DNAof 6 and 15 were obtained from premature infants P-32 and P-21, respec-

tively, and the value of 135 ,l. 02/30'/mg. DNAwas obtained from the adult A-29.

the premature infants and could be consideredinfinitely higher when only negligible activity waspresent in the liver of the premature infant.These data indicate the complete absence or onlyslight presence of tyrosine oxidation in livers ofpremature infants.

Table III shows the mean values for oxidativeactivity, based on liver wet weight, nitrogen,desoxyribonucleic acid and total liver activity persquare meter of body surface. The data showthat irrespective of the standard of reference usedfor oxidative activity, the livers from prematureinfants practically always showed minimal activityand they in no instance even approximated theactivity of the adult or full-term infant livers.

In Figure 1 the comparative effects of 500 pg.of ascorbic acid on the oxidation of tyrosine by thesoluble fraction of the liver of the premature in-fant and the adult are shown. It is clear that thisconcentration maximally stimulated the adult liver

54

4C

34SOftoch

ni Id" faction24

IC

*--sAdult with ascorbic acid (Ca.a.)x--xAduit withou a.a.

aC-aPrematu infant with c.a.4Prrnature infant without a.a.

DO--

O/

,, .~ . ~ .10 20 30 40 50 60TIME OF INCUBATION (MIN.)

FIG. 1. THE EFFEcT OF AscoRBic Acm ON THE Ac-TMTY OF THE TYROSINE OXIDIZING SYSTEMOF HUMANLIVm

but showed no effect on the activity of the solu-ble fraction of the liver from the premature in-fant. Similar results were obtained with concen-trations up to 5 mg. of ascorbic acid.

Table IV shows the effect of adding the super-natant fluid of boiled adult rat liver homogenateand the filtrate from the ultrafiltered rat liver solu-ble fraction to the soluble fraction of the liver ofthe premature infant. Neither of these additionsresulted in an increase in the oxidative activityof the liver of the premature infant. The addi-tion of the soluble fraction of the liver of the pre-mature infant had no significant effect on the oxi-dative activity of the adult rat liver indicating noinhibitory activity.

Table V represents values for the various inter-mediate compounds which resulted from the oxi-dation of tyrosine by the soluble fraction of pre-mature infant liver and adult liver. The adultliver selected, and used only in these observations,was obtained from an individual with carcinomaof the kidney and widespread metastases. As aconsequence of the pathological changes in theliver there was a decreased oxidative activity,28 ul. 02/30'/ml. (84 ,d. 02/30'/gm. wet wt.) com-pared to the previously noted average of 102 ,dl.0,/30'/ml. (308 pl. 02/30'/gm. wet wt.) for normaladult liver (Table II). This drastic reduction inoxidative activity in the pathologic adult livernevertheless resulted in a value which remainedconsiderably elevated above that of premature in-fant livers (84 pl. 0/30'/gm. wet wt. compared tothe previously noted average value of 25 pl. 02/30'/gm. wet wt. for premature infant liver-TableII). A pathologic adult specimen was used forthe analysis to approximate the activity of pre-mature infant liver and thus obtain minimal

240

DEVELOPMENTOF THE TYROSINE OXIDIZING SYSTEM IN LIVER

TABLE IV

The effect of additions of various fractions on tyrosine oxidation

Solublefraction

Subject Material added g1. 02/30'/ml.

I. Premature infantP-14 24P-14 Supernate of boiled soluble fraction of rat liver 15P-16 3P-16 Ultrafiltrate of soluble fraction of rat liver 3

Ultrafiltrate of soluble fraction of rat liver 0

TI. AdurltatAR-16 324AR-16 Soluble fraction of premature infant liver 294

biochemical differences. The biochemical values premature infant liver and .17 mg. per ml. forobtained with the premature infant liver were adult liver) and the presence of this substance wasmore significant since distinctly higher values confirmed in both instances by the subsequentwere obtained with the liver of an adult having formation and isolation of the 2-4 dinitrophenyl-had a disease which led to severe parenchymatous hydrazones. When other samples of the samedisruption. This approach again obviates the criti- tissue were incubated in air there was a trace ofcism that premature infant liver was more dis- 2-4 dinitrophenylhydrazone formed after the in-rupted by autolysis after death than normal adult cubation of the premature infant liver and noneliver. In this analysis (Table V) all of the after adult liver. These results, together withknown intermediates were determined with a com- the identity of the results obtained with the Briggs'bination of various methods. The concentra- reaction (.08 mg. per ml.) and with iodometrytion of p-hydroxyphenylpyruvic acid was deter- (.07 mg. per ml.), established that in the case ofmined following incubation of the soluble fraction the adult liver the Briggs' reacting material wasin an atmosphere of nitrogen (.04 mg. per ml. for all homogentisic acid. On the other hand, only

TABLE V

Protocol of a complete experiment using the soluble fraction of human liver

Premature infantliver Adult liver

Substance Under UnderDetermination determined In air nitrogen In air nitrogen

Oxygen uptake 1 0 28 0pl./30'/ml.

Millon test* Tyrosine + 1.52 1.55 1.47 1.64mg./ml. p-Hydroxyphenyl-

pyruvic acid

Briggs' test* p-Hydroxyphenyl- .08 .04 .08 .17mg./ml. pyruvic +

Homogentisic acid

Iodometry* Homogentisic acid .02 0 .07 0mg./ml.

Spectrophotometry* Maleylacetoacetic + - + +(X320) acid

Dinitrophenyl- p-Hydroxyphenyl- + - +hydrazones pyruvic acid

Nitrogen 4.67 4.25mg./ml.

* All determinations done after 60 minutes of incubation.

241

N. KRETCHMER,S. Z. LEVINE, H. MCNAMARA,AND H. L. BARNETT

a part of the Briggs' reacting material in the caseof the premature infant liver could be accountedfor by iodometry (.02 mg. per ml. leaving ap-proximately .06 mg. per ml. by difference). Whenthe premature infant liver and adult liver wereincubated in air, the end-product maleylaceto-acetic acid was detected in both cases. A greateramount of this substance was present when theadult liver was used. The lack of homogentisicacid (iodometry) and maleylacetoacetic acid(spectrophotometric analysis) when the sampleswere incubated in nitrogen proved the presence ofan anaerobic environment. Thus, by the compositeuse of various methods the various individualsteps in the oxidation of tyrosine were ascertained,and the differences between the premature infantliver and the adult liver were emphasized.

DISCUSSION

These studies show that the liver of the pre-mature infant has little or no tyrosine oxidizingactivity. The activity of the tyrosine oxidizingsystem is greater in the liver of the newborn in-fant and notably greater in the liver of the adult.These differences appear to be real and apparentlydo not depend on the conditions of obtainingspecimens as indicated by the rat autopsy modeland verified by the finding of a very low oxidativeactivity in the liver of the fetal rat (17).

The premature infants in this study wereyounger and smaller than the subjects in the pre-vious clinical studies (8) and the in vitro activityof fractions of their livers was not stimulated bythe addition of ascorbic acid or dichlorophenolin-dophenol. The addition of ultrafiltrates and boiledsupernates of adult rat liver did not activate theliver of the premature infant nor did the solublefraction of the liver of the premature infant in-hibit the activity of the liver of the adult rat.These experiments established the absence of non-protein inhibitors and the presence of the non-pro-tein activators in the liver of the premature infant.A highly significant feature of this study is thedemonstration that the activity of the tyrosineoxidizing system increased at a greater rate fromprematurity to adulthood than the concomitantincreases in cell nitrogen or for that matter anyother cellular standard of reference. The over-allrelationship of enzyme development to systemic

development and differentiation has been wellsummarized by Boell (26) and Needham (27)who have pointed out that the development ofsuch molecules as enzymes patterns morphologicdevelopment. Boell (26) states that enzymes withgeneral and vital functions apparently develop firstand keep pace with cell growth whereas enzymeswith more specific and less vital functions developmore slowly and later accelerate at a faster rate.

The data previously presented indicate that thelow activity of the tyrosine oxidizing system in theliver of the premature infant is not due to a lackof coenzymes or non-protein activators but to theactual absence of apoenzyme. Table V showsthat blockage with nitrogen gas at the first stepof tyrosine oxidation, using equivalent quantitiesof tissue nitrogen and an excess of a-ketoglutarateand pyridoxal phosphate, resulted in the produc-tion of considerably less p-hydroxyphenylpyruvicacid by premature (.04 mg. per ml. tissue) thanadult livers (.17 mg. per ml. tissue). This trans-amination was studied more completely in rats(17) and it was similarly observed that the tyro-sine transaminase apoenzyme was in much lowerconcentration in the liver of fetal rats than in theliver of adult rats. Transaminase activity maypossibly reflect the activity of each of the indi-vidual constituent enzymes of the entire enzymesystem.

No definitive evidence is at present available toexplain the apparent discrepancy between theseresults and the previous clinical observations (8,9). One may assume that early in the life of thepremature infant only a small amount of tyrosineis converted to p-hydroxyphenylpyruvate due tothe paucity of the tyrosine transaminase apoen-zyme. This small quantity of p-hydroxyphenyl-pyruvate would require only a meager concentra-tion of p-hydroxyphenylpyruvate oxidase for oxi-dation and the apoenzyme could be activated com-pletely by minimal stores of intrinsic ascorbicacid. The above discrepancy could then be readilyexplained by the further assumption that as thepremature infant develops more apoenzymes aresynthesized and extrinsic ascorbic acid is necessaryto activate the increased p-hydroxyphenylpyruvateoxidase apoenzyme. When this cofactor is notgiven to premature infants on a high protein diet,the tyrosine oxidizing system is blocked (8, 9) atthe stage of conversion of p-hydroxyphenylpyru-

242

DEVELOPMENTOF THE TYROSINE OXIDIZING SYSTEM IN LIVER

vate to homogentisate; and tyrosine and p-hy-droxyphenylpyruvic acid appear in the urine.Observations on premature infants are in progressto determine the in vivo and in vitro veracity ofthis theory.

The inconsistently beneficial effect of folic acidon the hydroxyphenyluria of premature infants(14), monkeys (13), and guinea pigs (12) maydepend on augmented nonspecific protein syn-thesis or even on synthesis of the specific apo-enzymes. In either case there would result agreater utilization by anabolism or catabolism, re-spectively, of the substrate presented, i.e., ty-rosine. The mechanism or mechanisms involvedin the reduction of tyrosyluria by the administra-tion of ACTHis being studied in animals.

SUMMARY

1. The activity of the tyrosine oxidizing systemof the liver of the human adult is 10 to 30 timesgreater, and the activity of the liver of the full-term infant 3 to 5 times greater, than the hepaticactivity of the premature infant. These markeddifferences were consistent, irrespective of thestandard of reference used.

2. The in vitro addition of large amounts ofascorbic acid did not stimulate the oxidative ac-tivity in premature infant liver in contrast to itsstimulating effect on adult liver.

3. Premature infant liver does not lack thermo-labile or thermostable non-protein activators nordoes it contain non-protein inhibitors of thetyrosine oxidizing system.

4. A protocol is presented in which each inter-mediate of the tyrosine oxidizing system wasstudied. The evidence suggests that the prema-ture infant in the first few days after birth lacksapoenzyme and that the apoenzyme subsequentlydevelops at a rate in excess of systemic cell ortissue growth.

REFERENCES

1. Knox, W. E., and LeMay-Knox, M., The oxidation inliver of L-tyrosine to acetoacetate through p-hy-droxyphenylpyruvate and homogentisic acid. Bio-chem. J., 1951, 49, 686.

2. Crandall, D. I., and Halikis, D. N., Homogentisicacid oxidase. I. Distribution in animal tissues andrelation to tyrosine metabolism in rat kidney. J.Biol. Chem., 1954, 208, 629.

3. Knox, W. E., The metabolism of phenylalanine andtyrosine in A Symposium on Amino Acid Metabo-lism, McElroy, W. D., and Glass, H. B., Eds.,Baltimore, Johns Hopkins Press, 1955, p. 836.

4. Lan, T. H., and Sealock, R. R., The metabolism invitro of tyrosine by liver and kidney tissues ofnormal and vitamin C-deficient guinea pigs. J.Biol. Chem., 1944, 155, 483.

5. Sealock, R. R., Goodland, R. L., Sumerwell, W. N.,and Brierly, J. M., The r6le of ascorbic acid in theoxidation of L-tyrosine by guinea pig liver extracts.J. Biol. Chem., 1952, 196, 761.

6. LaDu, B. N., Jr., and Greenberg, D. M., Ascorbicacid and the oxidation of tyrosine. Science, 1953,117, 111.

7. Williams, J. N., Jr., and Sreenivasan, A., Furtherstudies on the cofactors of the liver tyrosine oxi-dase system. J. Biol. Chem., 1953, 203, 605.

8. Levine, S. Z., Marples, E., and Gordon, H. H., Adefect in the metabolism of tyrosine and phenyl-alanine in premature infants. I. Identification andassay of intermediary products. J. Clin. Invest.,1941, 20, 199.

9. Levine, S. Z., Tyrosine and phenylalanine metabo-lism in infants and the role of vitamin C. HarveyLect., 1946-1947, 42, 303.

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