Atypical phenylketonuric heterozygote

T h e Journal of P E D I A T R I C S

351

Atypical p beny lketon u ric beterozygote D e f i c i e n c y in p h e n y l a l a n i n e h y d r o x y l a s e a n d t r a n s a m i n a s e a c t i v i t y

A child with an elevated serum level o[ phenytalanine, a typical phenylketonuria phenylalanine tolerance test, an absence o[ increased urinary concentrations of phenylketones, ortho- and parahydroxyphenyl acids, and phenolic acids, as well as no abnormality in urinary excretion of 5-hydroxyindoleacetic acid or in indole-3-acetic acid is reported. It appears that this child has marked limitation in phenylalanine transaminase activity and, as a consequence, the characteristic associated biochemical abnormalities related to limited activity o[ this enzyme were not expressed and could not be evoked.

John A. Anderson, M.D., Ph.D.,* Robert Fisch, M.D., Erma Miller, M.S., and Doris Doeden, M.S. MINNEAPOLIS, MINN.

T H E c L I N I C A L and biochemical features of the disease phenylketonuria ( P K U ) vary widely. The range of expression extends from those patients demonstrating the severe clinical and classical biochemical features to a small group of persons with transitory urinary findings ~ or only an elevated serum level of phenylalanine, typical homozygous plasma response to phenylalanine loading, and persistent absence, by ordinary methods, of increased urinary phenylketones or related characteristic metabolites. 2 Though it seems certain that these "atypical cases" are homozygous, the reports of them do not present studies concerning the possibility that some From the Department of Pediatrics, College of Medicine, University of Minnesota. Supported by the MeClure Metabolic Research Fund and the Minneapolis Association for Retarded Children. ~Address, Department of PedlatHos, University ol Minnesota Medical School, Minneapolis, Minn. 55455.

of these children may be heterozygous for the P K U gene. While phenylketones are usually not consistently present in the urine of P K U patients unless the serum phenylalanine is maintained above a level of approximately 15 mg. per 100 ml. (0.91 ~mole per milliliter), most of the milder cases do have associated urinary metabolites related to phenylpyruvic acid (PPA) and, hence, are most likely homozygotes. I t is well known that ortho-hydroxyphenylacetic acid (o.-HPAA) and para-hydroxyphenyllactic acid (p-HPLA) m a y be present in the urine in the absence of PPA when the serum level of phenylalanine is moderately elevated, indicating practically complete metabolism of PPA to these and other metabolites?, 4 O n the other hand, it is doubtful that patients with only an elevated serum level of phenylalanine without phenylketones in the urineor further metabolites of phenylpyruvic acid

3 52

March 1966

Anderson et al.

can be clearly defined as having phenylketonuria. Individual variations in the time of development of the clinical signs a n d symptoms and the time at which biochemical features are expressed have been noted during the development o f the disease in the first weeks of life. 5, 6 It appears that at this early stage the rate of development of enzymatic processes to the rising level of phenylalanine in the blood are quite variable, s Further, even in older institutionalized P K U subjects on the same diet, the level of the serum phenylalanine attained and the a m o u n t of PPA excreted also vary greatly. I n general, the distribution of the intellectual quotients found in P K U subjects demonstrating the milder biochemical abnormalities tends to be closer to the normal range, although exceptions have been noted in even mild biochemical expressions of the disease. 1 T h e present report is concerned with studies obtained on a 13-month-old female infant found to have an elevated serum level of phenylalanine, a typical P K U oral L-phenylalanine loading response for plasma tyrosine, normal intellectual and m o t o r development, and virtually complete and persistent absence of abnormal amounts of urinary phenylketones, o-HPAA, and metabolic derivatives of paxa-hydroxyphenylpyruvic acid ( p - H P P A ) . I t was also demonstrated that no disturbance in the excretion of indole-3-acetic acid, 5-hydroxyindoleaeetic acid, or of oxidative dissimilation products of t r y p t o p h a n formed along the kynurenine p a t h w a y was present either when the serum level of phenylalanine was elevated or when L-tryptophan loads were given. Phenylalanine loading tests in the parents revealed that the father was heterozygous and the m o t h e r normal for the P K U gene. MATERIALS

AND METHODS

The patient studied, J. K., was the third female child born of healthy patients. The first two children, ages 4 and 6, were healthy. Surveys of the father's-family, Irish-Norwegian descent, and of the mother's family, Italian descent, failed to reveal significant contributing information. The patient, whose birth weight was 7 pounds and 3 ounces, was found by routine Guthrie test-

ing to excrete an abnormal amount of phenyL alanine at one month of age. 7 A subsequent Guthrie test on the blood revealed 15 mg. of phenylalanine per 100 ml. (0.91 /~mole per milliliter). Confirmatory values on 2 samples taken at different times using the L-amino acid oxidase method s were 18 and 12.5 mg. per 100 ml. (1.13 and .76 /zmoles per milliliter), respectively. Repeated urinary tests for phenylketones using ferric chloride', Phenistix, and 2-4-dinitrophenyl-hydrazinc during the second and third months of life were all negative. Because of the persistent elevation in plasma phenylalanine, the child was placed on a low phenylalanine diet at 3 months of age and maintained on it to the time of the present study at 13 months of age when the child weighed 9.2 kilograms. During this time the motor, social, and adaptive responses were within normal limits. The Bailey Developmental Scale was found to be 8 to 10 months at 8 months of age and 13 to 14 months at 12 months of age. Electroencephalograms obtained at 12 and 13 months of age were entirely normal. During the time of low phenylalanine dietary control, plasma phenylalanine levels were 2.0, 1.5, and 0.8 mg. per 100 ml. at 4, 8, and 11 months, respectively. METHOD

OF S T U D Y

The patient, after having been on a low phenylalanine diet for 7 months, was admitted to the metabolic ward. After several days of adjustment on the same diet which contained only 25 mg. per kilogram per day of phenylalanine, 24 hour urine collections were made prior to, during, and after an oral tryptophan loading test. The child was thei1 discharged and an attempt to evoke excretion of phenylketones was made by placing the child on a diet (a general diet with added L-phenylalanine) containing approximately 250 mg. per kilogram body weight daily for a period of 3 weeks. Again, after a few days of adjustment in the hospital to essentially the same high phenylalanine diet, quantitative urine collections were made 24 hours prior to, during, and following the administration of a second oral tryptophan loading test. This was followed by an oral two dose L-phenylalanine loading test, and quantitative collections of urine were continued prior to and during the loading period. BIOCHEMICAL

METHODS

USED

Serum phenylalanine 9 and serum tyroslne 1~ were measured during the experimental study period. T o reflect changes in the me-

Volume 68 Number 3

Atypical phenylketonuric heterozygote

353

tabolism of phenylalanine via the tyrosine pathway or the PPA pathway as well as to reflect the possibility of inhibition of hydroxylation of p-HPPA, para-hydroxybenzoic acid (p-HBA) and its c~njugates, phloretlc acid (para-hydroxycinnamic acid), and coumarie acid (para-hydroxypropionic acid) and its glycine conjugates were measured by spectrofluorometric methods on the effluent of acidified urine passed through Dowex 50W-X12 (200-400 mesh) column. Two milliliters of the effluent were used to determine p-HPAA, coumaric acid, phloretic acid, and o-HPAA. This volume of effluent was agitated in 0.2 ml. of 0.25 per cent sodium nitrite for 3 minutes and then 0.2 ml. of 10 per cent ammonium sulfamate was added. T h e o-HPAA was extracted by adding 10 ml. of cold USP (Bakers) chloroform. An 8.0 mI. volume of the chloroform layer containing the o-HPAA was then removed and extracted into 2 ml. of KC1 borate, p i t 5.0, and the residuum was saved for coumarie, phloretic, and p-HPAA analyses. The chloroform layer was then removed and discarded, and the: amount of o-HPAA was determined in the KCI borate solution speetrofluorometrically by activating at 305 m/z and reading at 395 m#. The p-HPAA, coumaric, and phloretic acids were determined by reextracting the residuum of the original 2 ml. of effluent (from which the o-HPAA had been removed) with 8 ml. of either. Two milliliters of KCI borate; p H 5, were then added to 3.0 ml. of the ether extract and shaken. T h e ether was then removed and spectrofluorometric readings were taken, activating at 240 m/z and reading at 310 m~. This value reflected the content of both p-HPAA and phloretic acid; about 20 per cent of the fluorescence may be due to I-3-AA. It was then necessary to determine phloretic acid colorometrically using a modification of the method of Tompsett 1~ and correcting the value for phloretic acid from the value obtained speetrofluorometrieally for phloretic acid and p-HPAA. The amount of I-3-AA was measured by the method of Udenfriend,.independently, on another urine sample and this value was subtracted. T o determine coumaric acid, 3 ml. of the ether

extract containing p-HPAA and phloretic acid were mixed with 2 ml. of borate buffer at ph 9.9. The ether was then removed and spectrofluorometric readings were obtained on the borate buffer solution activating at 320 m/~ and reading at 440 m/x. Para-hydroxybenzoic acid and conjugates were determined by taking 10 ml. of the Dowex50W-X12 effluent and hydrolyzing in 4 ml. of 5 N HC1 for 20 minutes. Five milliliters of ether were then added to 2 ml. of the hydrolyzed effluent and agitated. Three milliliters of the ether extract were then evaporated in a clean test tube to dryness in a water bath at approximately 70 ~ C. This dried residue was redissolved in 2 ml. of warm distilled water and made basic by adding 0.1 ml. of 1 N NaOH. Readings were taken in the spectrofluorometer, activating at 295 m/z and reading at 350 m~. Urines containing at least 1 rag. per liter for each of the above phenolic acids could be measured with at least 85 per cent accuracy by the above procedures. Greater accuracy for p-HPAA and phloretic acid may be achieved by developing the color according to the Tompsett's 11 colorometrie procedure. It was noted that the color complex for p-HPAA produced by the 1-nitroso-2-naphthol in concentrated nitric acid fluoresces and the color complex produced by phloretic acid were negligible, contributing less than 5 per cent of the fluorescence. The material was activated at 450 m/z and read at 570 m/~ providing an accurate value for p-HPAA. Urinary catechol was determined by modification of the method by Booth 12 on the effluent of diluted acidified urine obtained from a Dowex 50W-X12 resin. Five-hydroxyindoleacetic acid was measured by the method o6 Udenfriend, Titus, and Weissbach. la Indole-3-acetic acid was measured by the method of Weissbaeh, King, Sjoerdsma, and Udenfriend. 14 Paracresols were measured by the method of Tompsett? 1 Kynurenine, acetylkynurenine, 3-hydroxykynurenine, anthranilic acid, and anthranilie acid glucuronide were measured by the coIumn chromatographic methods of Brown and Pric@ 5 and 3-hydroxyanthranilic acid was measured by the method of Tompsett? ~ Urines were

3 54

March 1966

Anderson et al.

tested for the presence of increased homogentisic acid using qualitative methods; detectable amounts were not found. RESULTS

After the child had been maintained on the diet containing approximately 2,350 mg. of phenylalanine daily for 21 days, an oral two dose L-phenylalanine loading test was done. A dose of 200 mg. per kilogram of body weight of L-phenylalanine was divided into two portions; one half was given initially and the other half 30 minutes later. Results obtained and presented in Table I indicated that the plasma tyrosine levels remained unchanged over a 5 hour period, indicative of practically complete absence of phenylalanine hydroxylase activity similar to that found in P K U subjects. Phenylalanine loading tests were done on both of the parents using tile two dose test described by Anderson and co-workers? 7 The results calculated as discriminant scores from the fasting, one hour, and two hour tyrosine values indicated that the father was heterozygous (discriminant score ---- 2.868) and that the mother was normal (discriminant score = 4.182). Values for normal subjects are 3.51 + 0.374, and for heterozygote subjects, 2.32 _+ 0.374 (Table I). Orthohydroxyphenylacetic acid is known to be excreted in large amounts (up to 400 mg. per day) in untreated P K U subjects, while normal subjects excrete only one to two milligrams daily. It may be found in phenylketonuric subjects who have moderately elevated serum phenylalanine when PPA is not detected in the urine by the usual methods. ~ It has also been found in the urine of P K U heterozygous subjects as well as in normal subjects when the phenylalanine in the blood is raised above a somewhat critical level of 9 to 11 mg. per cent (.54 to .67 /~mole per milliliter). 4 Evidence exists that PPA is the parent compound and that it is converted to o-HPAA by an oxidase similar in action to p-HPPA oxidase? s Nonspecific hydrox~;lation of phenylalanine has been reported producing both the orthoand the para- isomers of the hydroxyphenyl-

Table I. Plasma phenylalanine and tyrosine responses following oral administration of 200 mg. per kilogram body weight of L-phenylalanine as a two dose test. (Initial dose = 100 mg. per kilogram. Second dose, one half hour later.) Determination of presence of heterozygosity made by obtaining the discriminant score calculated from the tyrosine values using the formula: Discriminant score = 1.0 x logarithm of the fasting tyrosine v a l u e + 2.47 x logarithm of the one hour tyro.sine value + 4.15 x logarithm of the 2 hour tyrosine value. A discriminant score of 2.868 for the father and 4.182 for the mother indicated that the father was heterozygous and the mother was normal. (Mean values for normal subjects = 3.51 _+ .374, and for parent heterozygotes = 2.32 _+ .374) (Reference No. 17) Phenylalanine Time

Tyrosine

per #moles rag. per tLmoles r~Jo mI. per ml. 100 ml. per ml.

Child

Fasting 1 hour 2 hours 3 hours 3 ~ hours

10.0 27.8 30.8 39.3 51.8

0.60 1.69 1.88 2.39 3.15

1.8 1.8 1.6 1.9 1.8

.100 .100 .084 .105 .100

2.0 19.0 26.7 23.8 17.8

0.33 1.20 1.60 1.40 1.00

1.4 2.4 2.7 2.8 2.8

.08 .13 .15 .15 .13

1.2 18.0 22.2 18.5 17.1

0.21 1.09 1.33 1.10 1.00

1.4 3.5 4.4 4.0 4.0

.08 .19 .24 .22 .22

Father

Fasting 1 hour 2 hours 3 hours 4 hours Mother

Fasting 1 hour 2 hours 3 hours 4 hours

pyruvic acid 18, 20; however, the amount of o-HPAA produced appears to be relatively small. The results obtained in the present studies, Table II, indicated no change in the excretion of o-HPAA during periods when the serum phenylalanine was elevated by dietary means up to 18 mg. per cent (1.09 #moles per milliliter) or by oral loading with phenylalanine, increasing the serum phenylalanine level up to 51.8 mg. per cent (3.15

Volume 68 Number 3

Atypical phenylketonuric heterozygote

/~moles per milliliter). These observations provided further evidence that PPA, if formed, was not converted to o-HPAA or that no PPA was formed. While the results of the above study appeared to indicate absence of PPA and failure of formation of increased o-HPAA in the presence of added phenylalanine, it was of interest to inquire further into the excretion of phenolic acid metabolic products of tyrosine formed along a minor pathway that has been proposed by Booth and associates 19 (Fig. 1). Marked inhibition of para-hydroxyphenylpyruvic acid (p-HPPA) oMdase activity has been demonstrated when excess p-HPPA is present; PPA is also a powerful inhibitor. 19 Along this pathway p-HPLA and p-HPAA are formed from p-HPPA and are found in small amounts in the urine of rats under normal conditions, and in considerably larger amounts when excess tyrosine or pHPPA are given. P-HPPA is a substrate for the formation of p-HPAA and p-HPLA, and inhibition of p-HPPA oxidase activity results in increased production of these two para-hydroxylated phenolic acidsY ~ Accordingly, it was of interest in spite of the absence of urinap/ phenylketones in this subject to determine whether excretion of pHPAA and metabolites of p-HPLA would increase during phenylalanine loading. It appears that p-coumaric, phloretic, and phydroxybenzoic acid can be derived from p-HPLA according to the scheme of Booth. 19 Very slight increase in the total amount excreted of these phenolic acid derivatives, viz., p-coumaric, phloretic, p-hydroxybenzoic acid,

and p-HPAA occurred during phenylalanine loading periods. The sum of the excretions of these four metabolites of p-HPPA before loading was found to be 8.46 /,moles per hour, and during the loading period, 11.1 /xmoles per hour. Further, the excretion during the control period on the low phenylalanine diet was 9.0 /,moles per hour, and on the high phenylalanine diet, 9.1 /~moles per hour. It appeared that no inhibition of hydroxylation of p-HPPA could be demonstrated during the elevation of serum phenylalanine; hence, PPA or other inhibitors were most likely not formed. Contrary to the above observations, a reduction in the excretions of these four phenolic acids occurred in 2 institutionalized male P K U subjects (age 6 years, weight 19 kilograms; and age 10 years, weight 26 kilograms) when the plasma phenylalanine level was reduced by a low phenylalanine diet. When the plasma phenylalanine level in tile 6-year-old child was reduced from 1.9 /zmoles per milliliter to 0.44 /xmole per milliliter, the excretion of p-HPAA, phloretic, coumaric, and p-HBA and conjugates fell from 86.6 t~moles per hour to 36.4 t~moles per hour. An even more striking decrease occurred in the 10-year-old child when the plasma phenylalanine was reduced from 2.0/~moles per milliliter to 0.13 t~mole per milliliter. The excretion of these phenolic acids decreased from 105.8 to 35.6 /,moles per hour (24 hour specimens). Presumably these changes in the PKU children were related to a reduction in formation of PPA and further metabolites which are able to inhibit oxidation of p-HPPA.

L-(-)-tyrosine ....

~ p-hydroxyphenylpyruvic acid

p-hydroxyphenyllactic~enylacetic p . - c o u m ~" ~d< p-coumaroylglyczne

355

acid ~ phloretic acid

9 p-hydroxybenzoic acid

) p-OS03H-benzoic acid

Fig. 1. Minor metabolic pathway for tyrosine proposed by Booth and associates,a~ The underlined phenolic acids were quantitated in the urine of the subject, p-coumaroylglyeine was measured as p-coumaric acid, and the p-OSO,H-benzoic acid as p-hydroxybenzoic acid. (p-coumaric acid ~ para-hydroxypropionic acid; phloretic acid -- para-hydroxycinnamic acid.

356

March 1966

Anderson et al.

T h e excretion of urinary catechol was measured to determine whether increased amounts of phenylalanine could be disposed of by the formation of hydroxylated metabolites by a nonenzymatic formation of diphenolic compounds as has been suggested by Dalgliesh. 21 On the day when the fasting serum phenylalanine was .09 ~mole per milliliter, the excretion of catechol was .35 ~mole per hour after 3 weeks of the high phenylalanine diet. When the serum phenylalanine was .47 ~mole per milliliter, the catechol excretion was essentially the same, .4 ~mole per hour (Table I I ) . Further search of a pathway for the metabolic disposal of phenylalanine was made by measurement of the change in the excretion of paracresols on the possibility that phenylalanine may be converted to phenolic compounds which could be methylated and excreted as paracresols. It was of interest to note that, during the administration of the oral phenylalanine load, the paracresols in the urine increased from 0.6/~moles to 3.0 ~moles per hour. It has been shown that "nonspecific" hydroxylation of phenylalanine in both the position ortho- and parato the amino acide side chain can occur and that further hydroxylation ortho- or para-

to the new hydroxyl group can also occur. 2~ The fivefold increase suggested that some para phenolic compounds were formed, and that methyl substitution in the position parato the hydroxyl group occurred. Paracresol excretion in the 6 and 10-year-old institutionalized P K U subjects maintained on a general diet was found to be 38 and 58 /~moles per hour (24 hour sample), respectively, when the serum phenylalanine was 37 and 38 mg. per cent, and following treatment with the low phenylalanine diet, the paracresol excretions fell to 18 and 26/4moles per hour, respectively. T h e plasma phenylalanines at this time were 8.5 and 2.5 rag. per cent, respectively. In vitro studies indicate: that phenolic compounds are not formed by nonspecific hydroxylation of compounds containing the pyrrole nucleus, such as the oxidative dissimilation products of tryptophan, as the pyrrole nucleus is not attacked. 21 T h e failure of urinary paracresols to change during ntryptophan loading confirms this observation in vivo and suggested that tryptophan was probably not a parent substance for the formation of paracresols. It has been suggested that PPA and oHPPA inhibit tryptophan pyrrolase and, as

Table II. Urinary e x c r e t i o n of indole-3-acetic acid (I-3-AA), 5-hydroxyindoleacetic aclq radation pathway for tryptophan: kynurenine (Kyn.), acetylkynurenine (Ac. Kyn.), 3-hydrox~ and ortho-aminohippuric acid (O-AHA)

Time High phenylaIanine diet

Control period Phenylalanine loading

(hours) 10.0 5.5

I

2

3

gyn.

Ae. Kyn.

3-OH Kyn.

.6-3.15"

T T

T T

1.0 .58

.09t .13t

T .65

T .12

.33 1.17

.29 .9

.05 T

1.23

Serum phenylalanine

( #m01

(#moles per ml.)

Low phenylalanine diet

Control period Tryptophan loading

24 24

High phenylalanine diet

Control period Tryptophan loading

24 24

.47 1.04-1.09:~

T, Trace of metabolite. *Fasting and 5 hour value following phenylalanine load (Table I ) . tFasting A.Z~. value at beginning of collection period. ~Fasting and 6 hour value following tryptophan load; hourly values were similar.

.69

Volume 68 Number 3

Atypical phenylketonuric heterozygote

a consequence, reduce the excretion of metabolites which reflect the activity of tryptophan pyrrolase required for the formation of kynu renine.22 Hence, the quantitative excretion of the quinolinic intermediates along the tryptophan-kynurenine pathway was determined during the period when the plasma phenylalanine had been maintained at a low level, during a period when elevation due to increased dietary intake had occurred, and during the period of L-phenylalanine loading. The administration of an oral Ltryptophan load resulted in a change in the total excretion of the metabolites measured from .83 ~mole per hour before tryptophan loading, to 3.60 /~moles per hour during the loading period when the plasma phenylalanin had been maintained at a low level. During the period when the plasma phenylalanine had been raised to a higher level by a general diet, an oral L-tryptophan load produced a change from 1.50 txmoles per hour per day before loading to 3.64 /xmoles per hour during tryptophan loading. These responses to oral tryptophan loads are we.ll within the range of responses observed for normal children and previously reported from this laboratory? s T h e effect of the administration of excessive phenylalanine load on

the excretion of the tryptophan oxidative intermediates also failed to indicate interference in the rate of formation of kynurenine intermediates during the 5 ~ hour phenylalanine loading period (Table I I I ) . Failure to form PPA may explain these normal responses as it has been suggested that PPA may interfere with tryptophan-pyrrolase activity. 22 No abnormal response in excretion of 5HIAA or in I-3-AA occurred during the time when elevated serum phenylalanine levels were present, nor did administration of Ltryptophan load cause abnormal increases in I-3-AA. A competitive effect by phenylalanine on intestinal absorption of tryptophan resulting in increased I-3-AA excretion has been noted. 24 The usually reduced urinary excretion of 5-HIAA observed in untreated P K U subjects 25, ~6 was not found during the period when the serum phenylalanine level was elevated.

357

DISCUSSION

FSlling and co-workers27 reported the case of a phenylketonuric girl who excreted slight amounts of PPA, detectable only in the morning specimens. Cowie ~s and, later, Woolf and Vulliamy 29 reported cases in

(5-HIAA), and the following metabolites of tryptophan produced along the oxidative deganthranilic acid (3-OHAA), anthranilic acid (Anth. A.), anthranilic glucuronide (Anth. G.),

4

3-OHAA

per hour) .5 1.6

5 Anth. A. + Anth. G.

6

O-AHA

1-6 Sum of Kyn. metab.

I-3-AA

5-HIAA

T T

.16 T

1.76 2.14

(#moles per hour) .8 T 1.0 T

.5 1.28

T .33

T .25

.83 3.60

.65 1.7

.38 .84

T .42

.09 .25

1.50 3.64

.9 1.2

.8 .65 1.1 1.9

358

March 1966

Anderson et al.

Table I I I . Effect of administration of oral L-phenylalanine and L-tryptophan loads on the urina~ p-HPAA = para-hydroxyphenylacetic acid; p-HBA = para-hydroxybenzoic acid (including suil conjugate. The high phenylalanine diet contained 2350 mg. daily of phenylalanine; the low phenyl

High phenylalanine d i e t Control period Phenylalanine load

Period o[ collection (hours) 10 5.5

Low phenylalanine diet Control period L-tryptophan load

24 24

High phenylalanine diet Control period Trypto.phan load

24 24

PhenyIalanine (serum) (/Lrnolesper ml.)

Tyrosine (serum)

p-HPAA

2 Coumaric

2.0 2.O

4.2 2.6

1

.60-3.15"

.1-. 1*

.09t .13t

.035t .087t

1.3

.71

2.9 2.9

.087t .1,.I$

2.0 .16

5.0 3.7

.47t 1.04-1.095

T, Trace metahollte. *Indicates fasting and 5 hour serum phenylalanlne and tyrosine concentrations during loading period (Table I). ~'Indicates fasting A.ra. values at beginning of urine collection period. ~.Indicates fasting and 6 hour serum phenvlalanine and tyrosine coneentratlons during tryptophan loading period.

which PPA and its metabolites were only modestly increased and in which the plasma phenylalanine levels were: usually within the range of 7 to 10 mg. per 100 ml. (.43 to .06 /xmole per milliliter). In these cases, however, PPA and o-HPAA could be detected by more sensitive biochemical methods. More recently, Schneider and Garrard a~ have described a child with normal development who had hyperphenylalaninemia and intermittent phenylketonuria and wh% from loading tests on the parents, was found to be heterozygous. Hence, previously described cases with normal intelligence, phenylalaninemia, and transitory phenylketonuria, may not be true homozygotes. T h e child presented in this report appears to be heterozygous; however, the phenylalaninemia unassociated with phenylketonuria indicated the absence of phenylalanine transaminase activity. Recently Auerbach and co-workers al reported a premature infasat who demonstrated hyperphenylalaninemia, normal development, and absence of phenylketonuria, suggesting limited activity of phenylalanine transaminase. The phenylalanine loading responses of this child and of the parents indicated that the child was heterozygous. T h e absence of an increased excretion of PPA or other phenylketones, o-HPAA, p-

HPAA, and phenolic acid metabolic derivatives of p-HPAA presented evidence that PPA, was not available endogenously to serve as a parent substance for certain of the above metabolites, or as an inhibitor of hydroxylation of p-HPPA. These observations permit the conclusion that the enzymatic process for transamination of phenylalanine to PPA was virtually absent. At least one phenylalanine transaminase is known to be present in fetal liver and the formation of PPA appears to occur most rapidly in the presence of glutamine and glutamate? 2 Tyrosine alpha-ketoglutarate transaminase activity was likely present in this child as phenolic acid derivatives of p-HPPA as well as p - H P A A were present in the urine. Phenylalanine loading produced a slight increase (Table I I ) . In vitro studies indicate that tyrosine transaminase in the presence of alpha-ketoglutarate can convert phenylalanine to PPA. aa However, in spite of the in vitro observations, it appears that the tyrosine alpha-ketoglutarate transaminase did not act in presence of excess phenylalanine to produce increased urisaary PPA. It must be concluded that this heterozygous child demonstrated two defects in the metabolism of phenylalanine: (1) severe limitation in the conversion of phenylalanine to

Volume 68 Number 3

Atypical phenylketonuric heterozygote

359

excretion of hydroxyphenyl, phenolic and phenolic acid metabolites of phenylalanlne and tyrosine. fated conjugate); o-HPAA = ortho-hydroxyphenylacetic acid. Coumaric acid includes glycine alanine diet contained 230 mg. daily.

3 phloretic

4 p-HBA

Total o/ metabolites 1,2,3,4

l Catechol

f

per hour) .76 5.4

1.5 1.1

8.46 11.1

3.1 .68

1.7 1.8

9.0 6.1

.35 T

w w

2.1 2.1

9.1 3.4

.4 .4

Paraeresol

o-HPAA

(#moles per hour) .6 .16 3.0 .24 .5 1.0 .31 .4

.2 T .2 1.9

~Too l o w to m e a s u r e .

tyrosine, similar to that observed following loading in the homozygous subject, and (2) a severe limitation in phenylalanine transaminase activity. Indirect evidence of limited phenylalanine transaminafion activity was the absence of urinary phenylketones and metabolic products of them. Indirect evidence that PPA was not formed endogenously was provided by the studies of certain metabolic pathways for tryptophan. These included failure to evoke evidence of inhibition of tryptophan pyrrolase activity presumably due to PPA, failure to evoke increased excretion of I-3AA, failure to influence excretion of 5-HIAA, and failure to demonstrate a disturbance in oxidative metabolism of p-HPPA. This child was gradually removed from a low phenylalanine diet, and at 19 months of age the plasma phenylalanine level was slightly elevated at 5.6 mg. per cent, and at 27 months was 9.0 mg. per cent, T h e plasma tyrosine concentrations were 1.1 and 2.0 mg. per cent, respectively. A Stanford-Binet test revealed an intelligence quotient of 114 to 120 at 27 months of age. SUMMARY

Studies of an infant heterozygous for phenylketonuria revealed evidence of an elevated serum level of phenylalanine, failure

of serum level of tyrosine to increase following phenylalanine loading, absence of urinary phenylpyruvic acid and related metabolic products, and absence of secondary disturbances in the metabolism of tryptophan. The results of the studies indicate that two enzymatic defects may be present: (1) a deficiency in phenylalanine hydroxylase activity similar to that of the homozygote, and (2) a deficiency in phenylalanine transaminase activity required for the formation of phenylpyruvic acid. Normal development was present at 19 months of age. REFERENCES

1. Woolf, L. I.: Inherited metabolic disorders: Errors of phenylalanine and tyrosine metabolism, Advances Clin. Chem. 6: 97, 1963. 2. Woolf, L. I., Ounsted, C., Lee, D., Humphrey, M., Cheshire, N. M., and Steed G. R.: Atypical phenylketo.nuria in sisters with normal offspring, Lancet 2: 464, 1961. 3. Armstrong, M. D., and Shaw, K. N.: Studies on phenylketonuria: The excretion of o-hydroxyphenylacetic acid in phenylketonuria, J. Biol. Chem. 213: 797, 1955. 4. Cullen, A. M., and Knox, W. E.: O-hydroxyphenylacetic acid excretion in phenylalanine tolerance test for carriers, Proc/ Soc. Exper. Biol. & Med. 99: 219, 1958. 5. Armstrong, M. D., Centerwall, W. R., Horner, F. A., Low, N., and Well, W. B., Jr.: The development of biochemical abnormalities in phenylketonuric infants, in Folch-Pi, J., editor: Chemical Pathology of Nervous System, New York, 1961, Pergamon Press, pp. 38-50.

360

A n d e r s o n et al.

6. Allen, R. J., Heffelfinger, J. C., Masltto, R. E., and Tsau, M. U.: Phenylalanine hydroxylase activity in newborn infants, Pediatrics 33" 512, 1964. 7. Guthrie, R., Susi, A.: A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants, Pediatrics 32: 338, 1963. 8. LaDue, B. N., and Michael, P. J.: An enzymatic spectrophotometric method for determination of phenylalanine in the blood, J. Lab. & Clin. Med. 55" 491, 1960. 9. McCaman, M. W., and Robins, E.: Fluorimetric method for the determination of phenylalanine in serum, J. Lab. & Clin. Med. 59: 885, 1962. 10. Waalkes, T. P., and Udenfrlend, S.: A fluorometric method for estimation of tyrosine in plasma and tissues, J. Lab. & Clin. Med. 50: 733, 1957. 11. Tompsett, S. L.: The determination of volatile phenol in urine, Clin. Chem. 4: 237, 1958. 12. Booth, A. N., Robbins, D. J., Masri, M. S., and DeEds, F.: Excretion of catechol after ingestion of quinic and shlklmic acids, Nature 201: 191, 1960. 13. Udenfriend, S., Titus, E., and Weissbach, H.: Identification of 5-hydroxy-3-indoleacetic acid in normal urine and method for its assay, J. Biol. Chem. 216: 499, 1955. t4. Welssbach, H., King, W., Sjoerdsma, A., and Udenfriend, S.: Formation of indole-3-acetic and tryptamine in animals, J. Biol. Chem. 234: 81, 1959. 15. Brown, R. R., and Price, J. M.: Quantitative studies on metabolites of tryptophan in urine of dog, cat, rat, and man, J. Biol. Chem. 2195 985, 1956. 16. Tompsett, S. L.: 3-hydroxykynurenine in human urine, Clin. Chim. Acta 5: 415, 1960. 17. Anderson, J. A., Gravem, H., Ertel, R., and Fisch, R.: Identification of heterozygotes with phenylketonuria on basis of blood tyrosine responses, J. P~DIAT. 61" 603, 1962. 18. Taniguchl, L., and Armstrong, M. D.: Enzymatic formation of ortho-hydroxyphenylacetic acid, J. Biol. Chem. 238" 4091, 1963. 19. Booth, A. N., Masri, 1VI. S., Robbins, D. J., Emerson, O. H., Jones, F. T., and DeEds, O. H.: Urinary phenolic acid metabolites of tyrosine, J. Biol. Chem. 235" 2649, 1960. 20. Kirberger, E.: t)ber den abbau yon p-oxyphenylbrewztraubensaure bei leberkranken

March 1966

21.

22.

23.

24.

25. 26.

27.

28. 29.

30.

31.

32. 33.

unter dem einfluss yon 1-ascorbinsaure and anderen wirkstoffen, Ztschr. Physiol. Chem. 298: 245, 1954. Dalgliesh, C. E.: Nonspecific formation of hydroxylated metabotites of aromatic amino acids, Arch. Biochem. & Biophysics 58: 214, 1955. Tada, K., and Bessman, S. P.: Studies on tryptophan metabolism in oligophrenia phenylpyruvica, Pediatrica Japonica, Japanese Ped. Soc. 3: 41, 1960. Michael, A. F., Drummond, K. N., Doeden, D., Anderson, J. A., and Good, R. A.: Tryptophan metabolism in man, J. Clin. Invest. 43: 1730, 1964. Yarbro, M. T., and Anderson, J. A.: L-tryptophan metabolism in phenylketonuria, Proceedings of the Society for Pediatric Research, 34th annual meeting, 1964, p. 124. Pare, C. M., Sandler, M., and Stacy, R. S.: 5-hydroxytryptamine deficiency in phenylketonuria, Lancet 1: 551, 1957. Berendes, H., Anderson, J. A., Priggie, B., Ruttenberg, D., and Ziegler, M. R.: Phenylketonuria, Univ. Minnesota M. Bull. 29: 298, 1958. F6111ng, A., Mohr, O. L., and Rund, L.: Oligophrenia phenylpyruvica: A recessive syndrome in man, Skrifter Norske VidenskapsAkad. Oslo: Mat. Naturv. K., No. 13: 1, 1944. Cowie, V.: An atypical case os phenylketonuria, Lancet 1: 272, 1951. Woolf, L. I., and Vulliamy, D. G.: Phenylketonuria with a study of the effect upon it of glutamic acid, Arch. Dis. Childhood 26: 287, 1951. Schneider, A. J., and Garrard, S. D.: Persistent hyperphenylalanemia, Abstracts of American Pediatric Society, 75th annual meeting, 1965, p. 54. Auerbach, V. H., DiGeorge, A. M., Carpenter, G. G., and Dobbs, J. M.: Abstracts of Society for Pediatric Research, 35th annual meeting, 1965, p. 108. Kenneg, F. T., and Kretchmer, N.: Hepatic metabolism of phenylalanine during development, J. Clln. Invest. 38" 2185, 1959. Jacoby, G. A., and LaDue, B. N.: Non-specificity of tyrosine transaminase: An explanation for simultaneous induction of tyrosine, phenylalanine and tryptophan transaminase activities in rat liver, Biochem. Biophys. Res. Commun. 8" 352, 1962.