t
M5513.
149
Bn
79-339
Given
By
^
Theappearance ofa darkbluegreen
color
—
similar to the spot above—
whenferricchlorideis dropped in a
sampleof urineor ontoawetdiaper
suggests the presenceof
CHILDREN'S
BUREAU
PUBLICATION
NO.
388
•1961
an
inherited
metabolic
disorder
associated
^vith
mental
retardation
PHENYLKETONURIA
WILLARD
R.
CENTERWALL,
M.D.
SIEGRIED
A.
CENTERWALL,
M.D.
Departmentof Pediatrics, College ofMedicalEvangelists,
SchoolofMedicine,LosAngeles, California
U.S.
DEPARTMENT
OF
HEALTH, EDUCATION,
AND
WELFARE
Boston Public Library
foreword.
superintendent of DocumentsJUL
17
1961DEPOSITORY
Great strides
have
beenmade
inimproving
and
safeguardingthehealthofmothers
and
children ofthiscountry.The
risks of pregnancy
have been considerably reduced.Many
of the threats to thelivesof
newborn
babies have been removed,and
more
infants survivethaneverbefore.
Among
thosewho
sui'vive,however,aresome
who
are notfullyequippedto
grow
and
develop normally.At
birth,some
havedefectswhich
we
have been unabletoprevent.Correction
and
prevention of these defects is one of the most,,challengingfrontiers inmedicalresearch today.
This bulletin is a report of one such defect, phenylketonuria
While
itaffects onlya smallnumber
of infants born each year,ifun-treated, the condition results in thetragic destruction of the
mind
ofthe child, although he
may
have been born withnormal
mentalpotential.
Hopefully,
some
of theapproachesto screening,early detection,and
treatment described here for phenylketonuria can be applied tosimilar conditions
and
thus furnish clues for dealing with other inheriteddefects.
The
Bureau
isproud
topublish thiswork
by
Doctors Willardand
Siegried Centerw^all. Briefly, it introducessome methods
of detecting
and
managing
phenylketonuria,and
is addressed to public healthworkersand
physiciansingeneralpractice.KATHERINE
B.OETTINGER,
table
of
contents
NCIDENCE
AND
GENETICS
BIOCHEMISTRY
LINICAL
COURSE
SCREENING
PROGRAMS
6Mentally Retarded Populations 6
Well
Baby
Populations 8Other
High
Risk Groups 9TESTING
METHODS
. . . . - 9The Test-Tube Test With Ferric Chloride 10
The Diaper Test 11
The Phenistix Test 12
The Filter
Paper
Test 12The Dinitrophenylhydrazine Test-Tube Test 13
Serum
Phenylalanine Determination 14DIETARY
TREATMENT
15SUMMARY
20APPENDIX
A
21APPENDIX B
22APPENDIX
C
24BIBLIOGRAPHY
25PHENYLKETONURIA
PHENYLKETONURIA
(PKU)
holds a unique position inthefieldofmental deficiency. Itisuniquebecauseit iseasilydetected
and
when
diagnosed early the mental deficiency of this disorder canhe prevented or favorably modified
by
special dietarymanagement.
^'^The
histoiy ofPKU
datesbackto the early 1930's to a familyin
Norway
withtwo
retarded children.^The
mother
was
hauntedby
tlienotion thatthesechildren
had
a peculiar odor.Her
effortsto get help forher childrenand
tolearn thecause of thisodorled herfrom
doctortodoctor overa period ofyears. Finally sheinterested a Nor-wegianphysician
and
biochemist, Dr.Ashborn
Foiling.Inthe process of
examining
thechildren,Dr.Foilingdiscovered that the urinesofboth reactedwith ferricchloridetogivean
unusualgreencolor. This he proved
was due
tothepresence ofphenylpyruvicacid,
which
hewas
able to crystallize in pureform
from
the urinesamples. Itis
by
thesame
simpleferricchloride urinetestand
modi-fications thereof^'^^^ that early diagnosis of
PKU
is
made
possibletoday.
The
substancewhich
was
responsible for the peculiar odor of thetwo
childrenisoneof thebyproductsofphenylpyruvic acidintheurine,phenylaceticacid. Increased
amounts
alsooccurinthe sweatofPKU
patients,and
it is possible todetect this odor in the hair oronthe skin of such persons
who
have not bathed recently.The
odor hardly fails to bringsome
association to themind
of the personwho
first smells it. It has been variously described as a musty, horsy, or barnlike odor.
A
urinetest survey of severalhundred
patients intwo
nearbyinstitutions for the mentally retarded led to the discovery of eight
additional patients with this disorder including
two more
siblingpairs.
That same
yearDr. Felling'sdiscoverieswere
published in thethe abnormality
was
an inheriteden-or in themetabolism ofphenyl-alanine,
and
hecalledit"imbecillitasphenylpyruvica."INCIDENCE
AND
GENETICS
As
news
of thisnewly
discovered disease spread, patients ininstitutionsforthementallyretardedin
many
countriesweresimilarlytested.
Out
ofsuchstudiescame
figures forincidence, determinationsof type of inheritance,
and
a fairly complete clinical picture of thedisease.^
Study
of these patientsalsoprovided informationby which
much
of the metabolic error eventuallywas
mapped
out. Because phenylpyruvic acid, the substance responsible for the green color re-action with ferric chloride in the urine, is a phenylketone, thename
phenylketonuriaw^as suggested for thisdisease.^"
To
date, thisisthename
of preference.It
was
soon discovered thatPKU
isfound on
the average inone-half to1 percent of institutionalized mental defectives. Interpo
lating
from
this prevalence, it has been estimated thatPKU
occursonce in every 20,000 to 40,000 live births.^'" Sexes are essentially
equally affected
and
all racesappeartobeinvolved.However,
the in-cidence is relatively higher inpeople ofEuropean
stock^and
ispar-ticularly
low
inpeopleofJewish^^-^^orNegro
"-^^ancestry.Because
PKU
often involvesmore
than one child in a family,from
thebeginningitwas
recognized as a familialdisorder.^'®When
it
was
determined that the parents werenormal
and
thatapproxi-mately one in four childrenintheinvolved families
had
PKU,
itwas
concluded that
PKU
was
inheritedby
a simple autosomal recessivegene.®'^°
The
followingdiagram
represents themode
of inheritanceinaclassical
PKU
family (fig. 1).
In this situationthe
normal
parents are each carriers(Pp)
ofthe defectivegenefor
PKU.
The
childrenfrom two
suchparentscan be noncarriers(PP)
or carriers like the parents (Pp), or havetwo
abnormal genes (pp)and
have phenylketonuria(PKU).
Thus, each pregnancy of such parentshas a l-in-4: chance of resultingin aPKU
child (pp)and
a 3-in-4 chance of resulting in anormal
child(PPandPp).
If
we
assume that the incidence ofPKU
in the generalpopu-lation is 1 in 20,000,thenit can be determinedthat 1 in70of the
Figure 1,
—
Inheritance Pattern forPKU.
normal phenylketonuria
dividing 20,000
by
4and
then taking the square root.To
test thisfigureone can calculate inreverse that thechanceof
two
suchcarriersinthe generalpopulationto
marry
eachotherwould
be 70X
70 or oncein 4,900 (orroughly 5,000) marriages. If eachof these 5,000 couples,
including the "carrier" couple,
had
four children, then one of the 20,000offspringwould
beexpectedtohavePKU.
It shouldbe realizedthat exact incidence of
PKU
and
thus the incidence of carriers is as yetunknown.
This fact w^ill not bedeter-mined
until reports are gatheredfrom
wide-scale screeningpro-grams
*'^'''' of the general populationand
more
extensive use ismade
of
improved methods
for "carrier" detection.At
present each ofseveral proposed
means
of determining "carriers," i.e., phenylalaninetolerance tests,^" fasting
serum
phenylalanines,^'and
phenylalanine-tyrosine ration,^^ altliotigli useful, hassome
limitations in clinicalapplication
—
mainlybecause of overlapbetweennormal and
"carrier'*results.
BIOCHEMISTRY
Phenylketonuria is
known
asan
inborn error of metabolism.The
basicdefectcausedby
thePKU
gene islackof theparahydroxy-lase
enzyme which
normally changes the essentialamino
acidpheny-lalanine to tyrosine.^"'^"'^^
The
metabolicpathways
affectedby
thisenzyme
deficiency areshown
in thediagram
(fig. 2).Figure 2.
NORMAL PHENYLKETONURIA
OTHER URINARY
METABOLITES
OF PHENYLALANINE
PIGMENT OTHER METABOLIC METABOLISM PROCESSES
Becausethe
mother
hasessentiallynormal
metabolism,thebaby
is protected before birth.
As
soon as anewborn baby
withPKU
be-gins to take milk (breast milk or cow's milk), phenylalanine,
which
l>l()ekedin its
normal
metabolic pathway,the phenylalaninebuildsup
to
serum
levelsofabout20 times the normal.* Thistakes place fairlyrapidly, so that
by
the time the infant is 1 ^^to 6 ^ weeks of age, theibnormal byproducts of this high
serum
phenylalanine begin to ap-l^ear in the urine. It is believed that the continued high level of phenylalanine oritsrelatedmetabolitesisresponsible directly orindi-rectly for themental retardation. This viewis supported
by
the factthat low phenylalanine diets started
on
PKU
infants in the earlymonths
oflife, (sothatserum
phenylalanine levels remain within thenormal or near-normal range), has resulted in
normal
mentaldevel-opment.^-^
Many
questionsremainyetunanswered
aboutPKU
metabolism.Why,
forexample,istherelittleorno correlation between tlielevel of elt^vatedserum
phenylalanineand
tlie degree of mental retardation?^\hy
does a rare case ofPKU
now
and
then develop normallywith-out
any
treatment?What
role is playedby
the associated abnor-malityintryptophane metabolism? ^^CLINICAL
COURSE
The
untreatedPKU
patient is born apparentlynormal
but begins toshow
retardation early in life.At
about 3 to 4months
ofage, a subtle change
may
benoticed.The
infant beginsto loseinter-est in his surroundings,
and
thereafter development is sloweddown
orarresteduntil
by
2 or3years of age mostare inthe "below 50 I.Q."range ofmental deficiency.
(Of
theknown
untreated cases ininsti-tutions, 90 percent fall into this bracket^-^^ (fig.
3).) Occasional
untreated cases (perhaps 10 percent) are only mildly retarded
and
afew
cases (lessthanone-half percent) have beenreportedwithnormal
mentality.
-PKU
children usually arefairly well-developed physicallyand
haveno
trulydiagnostic stigmata (otherthanthe characteristicmusty
odor).
There
may
beslightstunting of heightand some
havesmallerthan average headsizes.
The
average age for sitting alone isfrom
12 to 15 months; the*If anewborn
PKU
infant doesn't receivemillc (proteins) the serum phenylalaninelevelstill will show some risewithin afew days due to catabolism ofbodyproteins.
average age for walking is 2i/^ years, for talking
3%
years.Some
never learn to walk;
many
never learn to talk.Approximately
80 percent are blonder than their parentsand normal
siblings. Be-havior patterns are frequently autistic, hyperirritable,and
destruc-tive.
About
80 percent haveabnormal
EEG's
and
approximately25 percent have convulsions. (Convulsions are
uncommon
after 10 years of age.)The
more
severely involved casesmay
show
signs ofupper motor
neurondamage
such as positive Babinskiand
ankleclonus.
About
25 percent ofPKU
patients have eczema,which
ispuritic
and
not limited toany
specific areas of thebody
and which
may
have itsonset within the firstfew
months
of life.^*SCREENING
PROGRAMS
Mentally
retarded populations
The
screening of mentally retarded populations forPKU
has been done formany
years in agood
number
of institutions for the retarded,^'®'^ whereas inmany
others such detection efforts are juststarting.^^
Although
it is unlikely that the intelligence of childrenpicked
up by
thismethod
can beimproved
verymuch
by
dietary control,diagnosingolde^'childrenissuchan important
way
of protecting subsequent siblingsand
other related infants withPKU
that routine screening is strongly reconimendecl in all institutionsand
schools orclasses for the retarded, both public
and
private.The
successofsuchprograms
willdepend
onmany
cooperatingorganizations. Case finding will have to be followed systematically
intothe
homes
of the familiesin away
somewhat
similarto screening familiesinwhich
an active caseof tuberculosis liasbeen found.A
recenthome
surveyof the siblings of20PKU
patientsataninstitution of 3,000 population uncovered three previously
undiag-nosedcases.-®
Two
ofthesewerestillyoung enough
to receive benefitfrom
treatment.On
the basis of this experience, severalrecommen-dations have been
made
for followupwork
with families ofPKU
patients.
1.
The
families should be carefully orientedand
counseled assoon as possible.
The
orientationshould also includean
Figure 3.
—
Clinical Gjurse in UntreatedPKU.
The
information should also be forwarded to the familyphysician.
At
least one informationpamphlet
for parentsis
now
available.^^2. All siblings should be screened as soon as possible, even if
the parents
do
not think they are retarded.Sometimes
re-tardation inyoung
children is unsuspectedby
the parents.3. All
newborn
siblings should have serirni phenylalaninedeterminations just prior to discharge
from
thenewborn
nurseries (at
two
ormore
days of age). If this isnot pos-sible, they should have urine testsat2, 4, 6, 8and
12weeks
of age.
As
an extra precaution,itisadvisabletodo
similarfollowup urinetests,evenon
newborn
siblingswho
havehad
a
normal
serum
level.so that
young
cousins, second cousins, nieces,and nephews
can betestedforPKU
duringearly infancy.Well-baby
populations
In
most
caseswhere
PKU
has been discovered inearly infancy,the diagnosis
had
beenmade
previously in a defective older sibling, as described above. If this werethe onlymeans by which young
in-fants withPKU
could be identified, itwould
mean
the sacrifice ofonechildineachinvolved family.
A
more
recently instituted typeof screeningprogram
involves routine screening testson all infants.*-^*' Thisis the onlyway
to findPKU
children in previously unidentified families earlyenough
for the infants to receivemaximum
benefitfrom
treatment. It is sug-gested that the test bedone
at the first three well-baby checkups.Phenylpyruvic acid usually does not appear in the urine until the
second or third
week
of life^^and
insome
cases not until 6 weeks.^For
this reason there isno
value intestingthe urine of babies in thenewborn
nursery.At
the present timemany
California healthdepartmentshave
joined in a routine screening
program
forPKU.
The
populationarea serviced
by
the participatinghealth departments is in excess of three-fourths of California.The
program would
not be possibleexcept for the full cooperation
and
enthusiasm of all of these healthdepartments.
The
test being used, called the diaper test, will be described later in this book.As
theprogram
is long range,no
finalresults can be reported at this time.
However,
it has already beenproved that routinetesting
by
thismethod
iseasily incorporated intothe regular well-baby program,
and
the simplicity of the test has helpedpromote
tlie enthusiastic support of theworkersin theclinics.Also ithas been provedthatthetestdoesuncovercasesof
PKU.*
Al-ready several
PKU
infants have been detectedby
routine screeningprograms
both in the well-baby clinicsand by
private practitioners.The
health departments of several other States have incoipo-rated this type of screeningprogram
into their well-child conferenceprograms.
Shortly after the diaper test detection
program was
initiated inCalifornia, a routinescreeningi)rogramwas
started independentlyin England. There it is advised that all babies be tested at about 1
month
ofageduringthefirst postnatal visit tothe clinic orhospital.The
screeningtestused isthe Phenistix®
Test-* as a urine or diapertest.* (See Testing Methods.)
New
cases are being discoveredby
routine screeninginEngland.®
In Cincinnati,Oliio, a screening
program
hasbeenstarted withthe so-called filter-paper test.^ (See Testing Methods.) In this
program, each
mother
witli anewborn
infant receives instructionsalono; with a piece of
hUer
paperand
a pre-addressed,stamped
envelope.
She
is instructed that,when
the infant is about 1month
old, thehker
paper isto be phiced in the baby's diaper mitil soakedwith urine. It is then removed, dried
by
open-air contact,and
sentvia mail to a medical center.
The
first 10,000 such tests has resultedinthediscovery ofa
young
infantwithPKU.
Other high
riskgroups
Because of the sometimes associated findings of behavior dis-turbances, convulsions, cerebral palsy
and
eczema inPKU
patients,it
may
be advisable to screen children presenting such problems inclinics,centers,or inprivatepractice, including thosewithor without mentalretardation.
TESTING
METHODS
**Several tests
and
modifications of tests arenow
available forthe identificationof
PKF.
A
studywas
recentlydone
atthe CollegeofMedicalEvangelistsSchoolof Medicine,
Los
Angeles,Calif.,totrytodetermineiftherewere
any
advantagesordisadvantagesofonetest over another for screening purposes. In this study several thousandevaluations were
made
on lo2 consecutive urine samples takenfrom
20 untreated institutionalized cases of
PKl\
The
eft'ects ofmany
factorson theurine tests were measured, such aspH,
specificgravity,turbidity, a.m.
and
p.m. voiding, ageand
sex of patients, houi-s ofstandingaftervoiding,etcetera.
The
most
important variablewhich
affected all the testswas
the freshness of the urine sample. Unless the urine has been frozen or a preservative added, it should be freshly voided for the bestre-sults (table I).
Although
deterioration is less rapidwhen
the urineis dried on filter paper, 10 percent will no longer test positive after
3 days. Factors such as cost of the test
and
ease of administrationwere also compared. All the tests studied were comparable in ac-curacy,
and
eachhad enough
specific meritstomake
that testvaluableundercertaincircumstances (tables I
and
II).••This section Is adapted from a paper in the November 1960 issue of the American JournalofPublicHealth=»with thepermission of theeditor.
It
was
foundtliat in each of thesetests the value is dependent onsome
simple but specific precautionswhich
if not understoodand
appreciated can easily cut the efficiency of that test considerably.
Most
ofthese pointers are included in the following discussion.On
rareoccasions
PKU
patientsfailtoexcretephenylpyruvicacid;there-fore, the possibility of this disease can not be excluded on the basis
ofone negative urine test. Because the diagnosis isof great concern
tothe family
and
involvesanexpensive,long-term treatment,all posi-tive urinetests should be confirmed with aserum
phenylalaninelevelbefore the diagnosis of
PKU
is establishedand
treatment begun.In a rare circumstance,
when
aserum
phenylalanine level can not beobtained within a week, treatment
may
be started on the presumptivediagnosis of
two
confirmatory urine tests, i.e., ferric chlorideand
dinitrophenylhydrazine. In such a case, however, a sample of
pretreatment
serum
should be frozen for later phenylalaninedetermination.
The
test-tube testwith
ferricchloride
The
test-tubetestwith ferricchloride isthe oldest, bestknown,
and
most widely used ofany
of the diagnostic urine tests for PKU.'''*' ^^The
color reaction of ferricchloride with phenylpyruvicacid is practically pathognomonic. Immediately there is a
medium-dark, blue-green to gray-green color Avhich fades in a matter of
sec-Figure 4.
—
Average Rate ofDevelopment
forUntreatedPKU
Children. i.Q. or D.Qonds, or minutes,depending
upon
the concentration of thephenylpy-ruvic acid inthe urine
and
tliestrength of the ferric chloride solutionbeing used. This fading of the color back to a neutral or negative urine color is most useful in dilTerentiating the true phenylpyruvic
acid reaction
from
most so-called false-positive reactions. Ingested salicylates, for example, will give the urine a blue-purple colorupon
ai^plication of ferric chloride. Unlike the phenylpyruvic acid
reac-tion, however, this color does not fade away.
Some
of the color re-sponses obtainedwhen
a ferric chloride solution isadded
to urinesamples are: green
from
bile, homogentisic acid (alcaptonuria), the catecholamines (pheochromocytoma),and
the urine ofmaple
syrupurine disease,^" red-brown
from
diacetic acid (acidosis), grayfrom
melanin (malignantmelanoma),
light violetfrom
chlorpromazine(Thorazine®)
ingestion,and
purplefrom
proclorperazine(Com-pazine
®
) ingestion. Colorreactions are fairlystableexceptwith ho-mogentisic acid. In this case the green color is extremely fleeting,disappearing withinasecondor two.
The
factthattheferricchloride reagent will demonstratethe presence of various drugsand
unusualmetabolites otherthan phenylpyruvic acid does not impair the values of thistest but rather
makes
itmore
interestingand
more
valuable.The
ferric chloride solutionisvery inexpensiveand
when
made
with distilled Avater
and
stored in polyethelene bottles it is stablein-definitely. "We have preferred a 10 percent solution because of its
quick, intense colorreaction.
When
afew
drops areadded
to a smallamount
(1cc.) ofurine,ithas beenfound
unnecessarytoadd
acidfirst because theferricchloride solutionisitselfveryacid(pH
1.8).
The
diaper
testThe
diaper test*-^^ is a modificationof the ferricchloride test-tube test.A
drop
of 10 percent ferricchloride is placedon
a baby'swet diaper (or even if the diaper has dried since being wet),
and
ablue-green to gray-green color appears immediately.
The
concomi-tant use ofa
drop
of acid isneither necessary nor desirable.As
withthe test-tubetest,the coloris transient.
Many
times adefinitelyposi-tive test will fade in less than half a minute.
Such
fading starts inthe center of the spot,
and
the last to fade is thegreen-rimmed
pe-riphery.
Whether
positiveor negative, the spotonthe diaperleavesapermanent
stain. This is perhapsworth
mentioning to the mother.Although
thousands of infants have beentested,we
havehad no
realcomplaints
from
themothers.The
same
false-positive reactionsoccur with the diaper test aswith the test-tube test.
The
test has proved to be inexpensiveand
simple to perform.
One
penny'sworth
of solution can test severalhundred
infants.Of
104PKU
urine specimens allowed to stand atroom
temperaturefor4 hours,97 (or93 percent) still definitely testedpositivewithferricchloride (tables I
and
II).
In practice it
was found
that in considerablenumber
of casesthe infant did not have a wet diaper,
and
themother
had no
wetdiaper with her.
For
this reason, mothers are instructed to bring along withthem
on the initialand
subsequent clinic visits the mostrecentlywetdiaper. In this
way
approximately30 percent additionalinfants
may
betestedwho
Avouldotherwisebemissed.The
Phenistix ®
^^'^^ testAnother
modification of theferricchloridetestisthePhenistix®
dip stick, a paper strip
which
is impregnated with a buffered ferricsalt. This reagent, according to our studies, is second only to
dini-trophenylhydrazine
(DNPH)
in sensitivity for phenylpyruvic acid.(See later discussion ofthe
DNPH
Test.)Of
104PKU
urines, 102(or 98 percent) still definitely tested positive with Phenistix
®
after 4hoursofstandingatroom
temperature (tablesIand
II). Thistest 1is excellent for routine screening of apparently well infants because:
the dip stick can be pressed against the still-wet diaper as well as
dipped into a urine solution. These sticks are stable almost
indefi-nitely if kept in their special container. False-positive reactions
do
occur with Phenistix'^but less frequently than with ferric chloride.
The
color reaction of Phenistix® with phenylpyruvic acid isessen-tially the
same
as the ferric chloride test.The
color alsomay
fadeaway
within a minute or so; thus, itwould
be impossible to place a Phenistix®
in a baby's diaperand
expect tocome
back laterand
getanaccuratetest result.
At
present the Phenistix®costs approximately 8 cents a test;thisis certainly inexpensive
and
should beno
barrier to those physi-cianswho
wish to utilize it as a screening test in their practices.Phenistix '^ is slightly
more cumbersome
than the diaper-test for routine screening;on
the otherhand, itleavesno
stain on the diaper.The
filterpaper
test "'^^The
filterpapertestisperformed
on ordinary white filterpaperwhich
has been wet withurine, dried,and
then sentthrough the mailto the testing laboratory.
The
test is thesame
as the diaper test.The
difference between the filter paper testand
the diaper test isthat the wet diaper
which
is tested is probablyno more
than several hours old, whereas the filter paper urinemay
be several clays old beforeitistested.The
question arises as tohow
long the dried, urine-soakedpaper continues to
^ve
an accurate test.To
evaluate this problem,30 urines from,
known
PKU
patients were soaked on filter paperand
dried to simulate the actual filter paper test.
Inasmuch
as thepro-gram
as setup
in Cincinnati involves experienced laboratoryperson-nel, even the faintest trace of positive reaction
was
considered as]30sitive.
By
the use ofthis criterion, the 30 specimensinitially wereall positive,but2 of
them
M-ereonlyvery faintly positive.By
3 days,at
room
temperature, the average time one can expect betweenvoid-ing
and
testingunder
suchaprogram, 3 (10 percent) of thetestspec-imens were negative.
At
5 days, 5 (16.7 percent) were negative.By
1 week, 6 (20 percent) werenegative. Theseresults indicate thatit
would
be a mistake to take one negative test as assurance thatno
PKU
ispresent. Ifimmediate
testing of a filter paper sample senttlirough the mail isnot possible, then store the sample in the freezer
until ready to be tested. Freezing arrests or
markedly
retards theprocess ofphenylpyruvic acid deterioration.
The
cost of materialsand
supplies in this screeningprogram
is approximately 1 cent per test providing the parents supply their
own
stamps on the envelopes.The
advantagesand
indications ofsuch a
program
are: (1) the screeningprogram
can be initiated inpractically all
young
infants (at least thosewho
are born inhospi-tals) ; (2) this test
might
be advantageous for useon
patients inout-of-the-way
and
distantplacesand
perhaps for infantswhere
there isno
availablewetdiaper for testingattheofficeorclinicvisit(tableII).
The
dinitrophenylhydrazine
test-tube testProperly prepared
and
used, the dinitrophenylhydrazine(DNPH)
29'31reagent isthemost
sensitiveand
reliable ofthe variousurine tests for
PKU.
False-negativetest reactions usingDNPH
on
known
untreated cases ofPKU
are rare (less than 1 percent).In
our experience with over 100 consecutive urine samples, therewere
no
false-negatives even after 18 hours of standing atroom
tempera-tures (tables I
and
II). Because other substances rarelyfound
inurine will react togive a positive test with the
DNPH,
any
positive testshould be cross-checkedwith aferricchloridetest.Of
theafore-mentionedsubstances
which
can giveacolorresponsewithferricchlo-ride, only the diacetic acid
and
the urine ofmaple
syrup urinedis-ease give positive reactions with
DNPH.
Thisreagentisprepared asfollows: about 4
grams
of2-4dini-trophenylhydrazine (an orange
powder)
areadded
to a liter of onenormal
hydrochloric acid. This mixtureisheatedina hotwater bath overnight tomake
a supersaturated approximately 0.3 percentsolu-tion of
DNPH.
The
supernatant, clear-yellow solution is filteredoff
and
stored in a dark glass bottle.Table I.
—
Percentage of 104PKU
Urine Samples Giving DefinitelyPositive Reactions After Increasing Intervals of Exposure at
Room
Temperature*
rases seemingly positive by urine tests.
No
infant or child shouldbestartedonthe long-term
program
of alow-phenylalaninediet with-out a blood sample being taken first.The
serum
specimen (2cc.)canbesafelystoredin afreezer
and
tested laterifan
immediateserum
phenylalanine determination is inconvenient or impossible.
In
thisway
there need beno
long delay in initiating dietary treatmentbe-cause oflocal lack of certain laboratory facilities.
Methods
for determiningserum
phenylalanines^^'^^'^*'^^ aretoo
complex
for description here.Many
medical school centersand
some
research centersand
commercial laboratories are setup
todo
thesedeterminations (see
Appendix
A)
.
There
aretwo
other indications for the use of theserum
phenylalanine level. Subsequent siblings born into a familywhere
PKU
is present in an older child have a 25 percent chance of alsohaving
PKU.®-
"
Newborn
infants with this disorderhave normal
cordblood phenylalanine levels; i.e.,under
5mg.
per 100 ml. serum.By
2 or 3 days postnatally, however, theserum
phenylalanine hasrisento abnormally high levels (10to 15 mg.),
and
a blood specimenat this timeis adequate to
make
the diagnosis.However,
it takes aweek
or longer (rarelyup
to 6 weeks) before theserum
level issuf-ficiently high so that phenylpyruvic acid can be detected in the
urine.^'"
Thus
it issuggested that blood specimens be takenon
thenewborn
siblingsofknown
casesofPKU
justpriorto theirdischargehome
from
thenewborn
nursery.^*' Babies in anewborn
nursery are tooyoung
to be urine tested forPRIJ.
It
might
be advisable to determine theserum
phenylalaninelevel
on
a childwho
seemsto suggest strongly the diagnosis ofPKU
but
whose
urine tests are negative.There
are instances ofPKU
in
which
serum
phenylalanine levels, although definitely elevatedabove normal,
were
just at the renal threshold level so thatspill-ageof metabolitesintothe urine
was
inconsistent.'®DIETARY
TREATMENT
Dietary- treatment for
PKU
was
first described in the early 1950's^^'^^'^^and
hassince been reported inenough
cases to establishits efficacy in the treatment of infants
and
small children.^'^"'*^'*^When
the diet has been started in the first severalmonths
of life, mental deficiency has apparently been prevented.The
rateand
reversibility of the deterioration are
somewhat
variable; however, it isgenerally felt that the dietmight
be tried on all children under 3years of age
and
thatmany
of thesemay
be significantly improved.The
cases of children above 3 years of age have to be consideredin-dividually. It is felt
by
some
workers in this field that olderPKU
children
who
areonlymildlyretarded orwho
havemarked
behavioral or convulsive disorders aremore
likely toshow
benefitfrom
dietman-agement
than others in thesame
age group.The
purpose of the diet is to lower the blood phenylalaninefrom
the abnormally high levels causedby
the disease (15-60mg.
per 100 ml. of serum) to near
normal
levels (1-3 mg.). Because all proteinsinnormally available foods are 4to 6 percent phenylalanine, it is impossible to devise a dietfrom
such foods that will lower thephenylalaninelevel
and
still providesufficient protein forgrowth
and
repair. Therefore, all
low
phenylalanine diets arebased on syntheticfoods
which
provideamino
acids with little phenylalanine.Most
ofthese synthetic foods have been
made
with a modified caseinhydro-lysate. Several products have been available commercially in the
United
States*^**and
Europe.^^*^ Actually, these special productsare so low in phenylalanine that if given
by
themselves theywould
produce a phenylalanine deficiency, including poorgrowth
and
a paradoxical rise inserum
phenylalanine due to catabolism ofbody
protein. Phenylalanine is an essential
amino
acidand
thebody
re-quires a certain
minimal
daily amount.*^-*^'*^An
opportunity isthus provided for varying the dietby
the addition oflow
proteinvege-tables
and
fruits—
withthe goal ofmaintaining aserum
level offrom
2 to 6
mg.
of phenylalanineper 100 ml. of serum. (Thesearebelievedto besafelevels.
How
high thephenylalanine can be keptand
still get the best results has not been determined.)The
serum
level ofphenylalanine at
which
phenylpyruvic acid will begin to appear inthe urineisbetween10
and
15mg.
per 100ml.^®Thus
frequent urine testsaregood
asgross checks of dietarycontrol,but they donot sup-plant the need of periodicserum
level determinations. It is advised that theseserum
levels be obtained at intervalsnot lessthan every 2to 4
months
while a child is on thelow
phenylalanine diet (andmore
frequently duringthe early
months
of control).The
serurtilevels arenecessary to detect both excesses
and
deficiencies of phenylalanine sothe dietcanheadjusted accordingly.
Our
experiencetodatehaslargelybeen withanAmerican-made
product, Lofenalac®.** Lofenalac® has fat, carbohydrate,
and
cer-tain mineralsand
vitamins incorporated with the low phenylalaninecaseinhydrolysate.
One
measure
(tablespoon) ofLofenalac® powder
has approximately 11/^
gm.
of protein equivalent, with 71/2 i^ig- of phenylalanine.One
measure (tablespoon)added
to 2ouncesofwatermakes
a 20 calorie-to-the-ounce formulawhich
has almost thesame
consistency,appearance,
and components
as milkminus
90 percent of its phenylalanine. It has a nut-like flavorwhich
has been wellac-cepted by almostall ofourpatients.
Most
of the children have takenit as a beverage either standard strength or
somewhat
concentrated. Several have preferredtotakeitmixed
directly into their lowprotein food supplements.The
powder
can also be utilized in speciallow
{)henylalanine recipes for pastries, breads, puddings, ice creams,
and
sauces.^°•'^^
Realization
on
thepart ofthe mother, or othersresponsi-blefor feedinga child,of
how
thedietcan bevariedand
stillmeet
thechild'sneedswillleadto easiercontrol
and
successwiththediet. Quitenaturally it is easier to introduce this
new
diet to a smallbaby
thanto anolderchild.
In the early
months
of life,we
supplement the baby'slow
phenylalanine formula with a small quantity of milk plus extrayita-inins
and
iron.As
thebaby
gets olcier, all varieties of fruitsand
certainlowproteinvegetables (i.e.,carrots, beets,string beans, squash,
turnips,tomatoes, etc.) are added. These low proteinfoods continue
to
form
the basis of the food supplementsand
later,depending on
the
amount
of phenylalanine allowedand
the child's preference,two
or three small servings of cereal, potato or cookies
may
beadded
tothe daily
menu.
With
all our patients on low-phenylalanine diets,we
continue to give small doses of supplementary vitaminsand
ironas an extra precautionaiy measure. Actually with the exception
of vitamin
C
the Lofenalac® Formula
is probably adequate in thesefactors. Several sample diets for infants
and
young
children aregivenintable III.
From
the samplemenus
in table III,you
will notice ahandy
rule of thumb. Children, about 1 year of age, need approximately
1 measure (tablespoon) of Lofenalac
®
perpound
ofbody
weight eachday.
For younger
infants theneedisproportionatelygreater,and
for olderchildren,itisproportionately less.^- Thisplus the daily calorieand
phenylalanine requirements is outlinedmore
graphically intable IV.
One
of themeans
of helping parents is to providethem
thefood lists
and
special recipes that have been devised forlow-phenyla-lanine diets.^"'^^
The
food lists give the equivalentamounts
of foodthatprovide15
mg.
of phenylalanine,calledone"equivalent"accordingto a plan devised
by
Lyman.^^By
substituting equivalents ofvari-ous foods, the parents can easily vary the diet
and
still keep withinthe prescribed
amount
of phenylalanine. (SeeAppendix
B
forex-change lists for low-phenylalanine diet.)
We
do
notmean
to suggest that every childwill taketo alow-phenylalaninedietwithout
any
difficulty.Depending on
his personal-ity,the older the childis, themore
hewill misshis previous diet,and
the
more
resistance he will putup
to thenew
diet.But
in ourn
s
C/5
experience, the difficulties^^'^* that were encountered with older
mix-tures (i.e.,diarrhea,starvation, severe weightloss, hypoglycemia,
and
convulsions)
have
notbeen a problem.Within
aweek
even 2and
3year olds have been drinking the Lofenalac
®
and
liking it—
and
without the use of special flavoring or sweetening.
We
agree with I'mbarger^^ about the disadvantages of hospitalization. All of ourchildren
were
started on the diet athome
and none have
required hospitalization because of the Lofenalac®
diet.Public health nurses
and
nutritionists are valuable assistantsin this field of diet
management.
However,
we
feel thatany
physi-cian (not blessed with suchhelpers)
who
is able to spendsome
extratime with the family at the start can supervise this diet.
Conscien-tious continued followup guidance
and
counseling are essential forlong-rangesuccessoftheprogram.
Table IV.
—
Some
Basic Daily Requirements of theLow
Phenylalanine DietandSuggestedQuantities ofLofenalac®Agegroup Totalcalories perpound ofbody weight Total phenyl-alanineper poundof bodyweight (milligrams) Measuresof Lofenalac® perpound ofbody weight (table-spoons) 0-3 months. . 3-12 months 1-3 years... . 3-7years 60-65 55-60 50-55 40-50 20-22 18-20 16-18 10-16 1-1ji
M-i
The
length of time that a child should be kept on the diet toobtain optimal
and
pennanent
benefit has been the subject ofmuch
interesting discussion. In the natural course of the disease, it seems
that deterioration does not continue
much
beyond
3 years of ageand
thishas been quoted as the age after
which
the special dietmay
not be necessary.The
issue has been confusedby
the fact that afew
PKU
childrenwho
were not treated until after 3 years haveshown
good
improvement.At
the present time, scattered experiences are indicating that children over 3 are maintaining their I.Q.'s off the diet.However,
since it seems to be true that the adverseperson-ality effects of phenylketonuria as well as mental deficiency respond
somewhat
to the low-phenylalanine diet, it will be interesting to see whether this will be a factor in the duration of treatment. Itwill take a
few
more
years to be certain of the longterm
effects ofdiet treatment
on
both mentalityand
personality.The
costof thesespecialdietaryproductsisofpracticalinterest.Manufacturers preparing these products as nonprofit service items are
making
every effort to supplythem
at as low a cost as possible.At
present*the dietaryproduct can probablybeprovidedtotheparentsthroughthe
pharmacy
atfrom
$0.75 to $2.00 aday,depending
on the sizeof thechild.It should be realized that the child
must
beunder
a doctor'scare at all timeswhile on the diet to assure the maintenance of
pre-scribedlevelsofphenylalanine bothfor
maximal
benefit tothechild'smentality
and
toprevent dietary deficiency.SUMMARY
Ithas been
shown
tliatthementaldeficiencyofphenylketonuria(PKU)
can be prevented or favorably modified if a special low phenylalanine diet is started early in life.Those
concerned withchild health are faced with the challenge of finding these children
during infancy.
The
history of the disease is reviewed brieflyand
theclinicalpicture
and
biochemical abnormalities aredescribed.The
various facets of the problem of case findings are discussed under
screening of mentally retarded populations
and
screening of wellbabies. It is pointedout that theidentification of the retarded child
with
PKU
isof importance atany
age because the family can thenbe alerted tothe possibility ofthisconditionina
younger
siblingwho
could be treated.
The
screening of well babies is important as the onlyway
of finding thefirstcase in afamily in timeto preventmentaldeficiency.
Tests usedin diagnosing
PKU
are describedand
evaluated for the purpose of choosing the appropriate testand
applying it withthe
most
effectiveness.Dietary treatment isdescribed
and
general principles forpre-scribing the diet for various ages are outlined. It is suggested that
all children
under
3 yearsof agediscovered to havePKU
(andsome
selectedchildren
beyond
thisage)might
be givenatrialof treatment.Ithasnot yetbeen established
how
longthespecial diet willbeneces-sary.
The
actualupper
limitfor theserum
phenylalaninelevelwhich
will permit
normal
development isunknown,
but it is suggested that 20the levelsbe keptwithintherangeof2to6
mg.
per 100ml.Examples
ofdailydietsare included.
acknowledgments
We
acknowledge the valuable assistance given to usby
Mrs.Phyllis B. Acosta, M.S., Assistant Professor, College of Medical
Evangelists School ofDietetics,
Los
Angeles, Calif., inpreparingthesection on Dietary
Treatment and
to Mrs.Ada
W.
Turner, B.A.,Editor of Official Publications, College of Medical Evangelists, in
giving manuscript suggestions.
The
illustrations used in this paperare adapted
from
a slide lecture on phenylketonuria in theLoan
Library of
Mead
Johnson
&
Co.^*'APPENDIX
A
Some
Laboratory
FacilitiesWilling
To
Perform
Serum
Phenylalanine Determinations
As
away
of providingsome
immediate assistance to personshaving
no
local facilities available toperform
serum
phenylalaninedeterminations, several medical centers in the
United
States doingresearch in this field have been approached
and have
expressed a willingnesstoperform
such determinationssenttothem.The
follow-ingnames and
addressesmight
beusedforsuch purposes:
Armstrong,MarvinD.,Ph.D.
TheFelsResearchInstitute YellowSprings, Ohio Berry,HelenK.,M.A. Guest,GeorgeM.,M.D. TheChildren's Hospital EUand Avenue andBethesda
Cincinnati29,Ohio
Jervis,GeorgeA.,M.D.
LetchworthVillage
Thiells,Rockland Co.
New
YorkLaDu,BertN.,M.D.
Department of Health, Education, and
Welfare
NationalInstitutesofHealth Bethesda 14,Md.
Garrisi,JosephA.,M.D. Departmentof Pediatrics
College ofMedicalEvangelists
1720 Brooklyn Avenue
Los Angeles33, Calif.
Hsia,DavidY.,M.D. DepartmentofPediatrics
TheChildren'sMemorialHospital
707 FullertonAvenue
Chicago 14, 111.
O'Brian,Donough, M.D. DepartmentofPediatrics
University of Colorado Medical Center
4200EastNinthAvenue
Denver 20,Colo.
This isadmittedly an incomplete listof places able
and
willing;to
perform
such services.As
indicated, it is published here only asa
way
of suggesting afew
resourceswhich
are willing to help untillocalresources are located or
become
available.APPENDIX
B
(Reprinted with permission from: Phenylketonuria: DietaryManagementbyPhyllisBrownAcosta
andWillard R.Centerwall,in theJournalof theAmericanDieteticAssociation,Vol. 36,No.3, March,I960,p.207.)
Exchange
lists forlow
-phenylalanine dietFOOD
AMOUNT
ListI—
Lofenalac 30Mg.Phenylalanine—
2 Equivalents* Lofcnalact (dry) 4tbsp. Lofenalac (reconstituted) 1 c. ListII—
Vegetables 15 Mg.Phenylalanine—
1 Equivalent Beans,greenStrainedandchopped V^/itbsp.
Regular 3 tbsp.
Beets
Strained 2tbsp.
Regular 3tbsp.
Cabbage, raw,shredded 4tbsp.
Carrots
Strainedandchopped 3 tbsp.
Raw \ilarge
Canned 4tbsp.
Celery,raw 1)^small
stalks
Cucumber,raw Yzmedium Lettuce,head 2 leaves Spinach, creamed
—
strainedand chopped 1)^ tbsp. Squash Winter Strained 3 tbsp. Chopped 6tbsp. Cooked 2tbsp. Summer,cooked 4tbsp. Tomato Raw. \ismall Canned 2tbsp. Juice 2}.^tbsp.
List III
—
Fruits15Mg.Phenylalanine
—
I Equivalent Banana 4tbsp.Dates, dried 2 Fruitcocktail,canned lYi tbsp.
Grapefruit Sections Yzc. Juice Ysc. Orange Sections 3 tbsp. Juice 3 tbsp.
Grapejuice Ysc.
Lemonjuice 3 tbsp.
Peaches
Raw Ysmedium
Cannedinsirup IY2halves Strained 5 tbsp.
Chopped 7tbsp.
Pears
Raw Yimedium
Cannedinsirup 3halves Strained andchopped 10tbsp.
Pears and pineapple, strained
and chopped 7 tbsp.
Pineapple
Raw
YsC-Cannedinsirup 1}^smallslices
Juice )^c. Plums, cannedinsirup 1/4medium
Plums withtapioca
Strained 5 tbsp. Chopped 7tbsp. Prunes Cooked 2medium Juice Kc. Strained 3tbsp. Raisins l}itbsp. Strawberries 3large Tangerine Yssmall Watermelon %c. ListIV
—
Breads 30Mg. Phenylalanine—
2 EquivalentsBarleycereal,Gerbcr's, dry,.. lYztbsp.
Biscuits t 1small
Cereal food, Gerbcr's, dry.... 2tbsp.
Cookies,arrowroot l}i
Corn 2tbsp.
Cornflakes )^c. Crackers
Barnumanimal 6
Saltines 3
CreamofWheat, cooked 2tbsp.
Farina,cooked 2}itbsp.
Mixedcereal,Pablum,dry.... 1%tbsp.
Oatmeal
Gerber's strained 1%tbsp.
Pablum,dry 1^tbsp.
Potatoes,Irish IY2tbsp.
Rice Flakes,Quaker }^c.
RiceKrispies,Kellogg's )^c.
Rice, Puffed,Quaker
J.^c.
SugarCrisps )^c.
Sweetpotatoes oryams
Cooked 3 tbsp.
Strained 4tbsp.
Wafers, sugar,Nabisco 6
Wheat, Puffed,Quaker J^c.
List
V
—
Fats 5 Mg. Phenylalanine—
1/3 Equivalent Butter 1tsp. Cream, heavy 1 tsp. Margarine 1tbsp. Mayonnaise 1}^ tbsp.Olives,ripe 1large
ListVI
—
Desserts 30Mg. Phenylalanine—
2 Equivalent Cookies Riceflour 2 Corn starch 2 Icecreamt Chocolate ^^c. Pineapple ^ic. Strawberry ^^c. Vanilla.'H
c PuddingsJ 1c.Sauce,Hershcysirup 2tbsp.
List VII
—
FreeFoods; LittleorNo
Phenylalanine;
May
Be UsedasDesiredCandy Butterscotch Creammints Fondant
Gum
drops Hard Jellybeans Lollipops Cornstarch Guavabutter HoneyJams,jellies,and marmalades. Molasses
Oil
Sauces Lemont Whitet Sirups Corn... Maple.. Sugar Brown.. White. . Tapioca...
ListVIII
—
FoodstoAvoid;HighPhenyl-alanine Content;
May
Be Used OnlyOccasionally in Very Small Portions
Breads,most
Cheeses ofallkinds
.
Eggs
Legumes,dried
Meat,poultry,fish..
Milk
Nuts
Nutbutters
tSpecial recipemustbeused. *Oneequivalent maybedefinedas providing
ifMilkishigh in phenylalanine(1 oz.contains 15.mg.phenylalanine.
50mg.),butitmaybeordered in infants tokeep fMeadJohnson&Company,
phenylalaninebloodlevelsuptonormal.
APPENDIX
C
SuggestedReadingforGeneral
Review
ofPhenylketonuriaJervis, G. A.: Phenylpyruvic Oligophrenia (Phenylketonuria), A. Res. Nerv.
&
Ment.Dis.Proc.33:259-282,1954.Wright, S.W.,andTarjan,G.: Phenylketonuria,A.M.A. J. Dis. Child.93: 405-419 (April) 1957.
Knox,
W.
E.,and Hsia, D. Y.: Pathogenetic Probletns in Phenylketonuria, Am. J.Med.22:687-702 (May) 1957.
Lyman, F. L.: Phenylketonuria,
New
YorkJ. Med. 58: 3653-3656 (Nov. 15) 1958. Centerwall,W.
R.: Phenylketonuria,A
General Review, J.A.D.A. 36: 201-205(March) I960.
Literature forLayPersonsandParents
Brecher, R., and Brecher, E.: The Conquest of Body Chemistry Diseases, Family
Circle,October, 1957.
Centerwall,S.A.,and Centerwall,
W.
R.:An
Introduction toYourChildWho
has Phenylketonuria, (7 pages),College ofMedical Evangelists Press, Loma Linda,California,1958.
Brecher, R., and Brecher, E.: Saving Children from Mental Retardation,The
Sat-urdayEveningPost,Nov.31,1959.
BIBLIOGRAPHY
1. Horner, F.A., and Streamer, C. W.: EfiFect of a Phenylalanine-Restricted Diet on Patients with Phenylketonuria; Clinical Observations in Three Cases.
J.A.M.A.,161: 1628-1630. 1956.
2. Knox,
W.
E.:An
Evaluation ofPhenylketonuria with DietsLow
inPhenylala-nine. Pediatrics,26:1-11. 1960.
3. Centerwall,
W.
R.,and Centerwall, S.A.: Phenylketonuria (Polling's Disease):TheStory ofIts Discovery. J.Hist. Med. and AlliedSci. (In press).
4. Centerwall,
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16. Hsia,D. Y.,DriscoU, K.W.,Troll,W., and Knox,
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17. Knox,
W.
E., and Messinger, E.C: The Detection in the Heterozygote of theMetabolic Effect of the Recessive Gene for Phenylketonuria. Am. J.
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18. Hsia, D. Y.: Phenylketonuria: The Phenylalanine-Tyrosine Ratioin the
Detec-tion of the Heterozygous Carrier. J. Ment.Defic. Res., 2: 8-16. 1958.
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&
Med.,82:514-515. 1953.20. Udenfriend, S., and Bessman, S. P.: The Hydroxylation of Phenylalanine and
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21. Kaufman,S.:TheEnzymicConversion ofPhenylalaninetoTyrosine. Biochem.
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22. Armstrong, M. D., Centerwall,
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B.: The Development of Biochemical Abnormalities in PhenylketonuricInfantsin Chemical Pathologyof the Nervous System.
New
York: Pergamon Press. (In press).23. Pare C. M., Sandler, M., and Stacey, R. S.: 5-Hydroxytryptamine Deficiency in
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24. Paine, R. S.: The Variability in Manifestations of Untreated Patients with
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26. Centerwall,
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