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HYPERPHENYLALANINEMIA AND MENTAL RETARDATION
THE EFFECTS OF A HIGH MATERNAL PHENYLALANINE
BLOOD CONCENTRATION ON MOUSE OFFSPRING
i
APPROVED*
,•1 i• /. j,1
Majo^' Professor T
Minor Professor /
Dean of the School cgF Equation
Dean of the Graduate School
HYPERPHENYLALANINEMIA AND MENTA'L RETARDATION i
THE EFFECTS OF A HIGH MATERNAL PHENYLALANINE
BLOOD CONCENTRATION ON MOUSE OFFSPRING
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF ARTS
By
Stephen A. Mozara, Jr., B, A,
Dentoni Texas
January, 1970
TABLE OF CONTENTS
Page
LIST OF TABLES iv
Chapter
I. INTRODUCTION v
II. METHOD 17
Subjects Apparatus Procedure Intubations
III, RESULTS 24
IV. DISCUSSION 29
V. SUMMARY AND RECOMMENDATIONS 38
Summary
Recommendations
APPENDIX ^
BIBLIOGRAPHY
iii
LIST OF TABLES
Table Page
I, Total Percent of Surviving Offspring in Each Group . . . . . . . . . . 2k
II. Percentages and Levels of Significance Between C and E Groups . . . . . . . . . 2?5
III. Percentage and Levels of Significance
Between E and E Groups . • • 26
IV. Group Mean Maze Errors Per Day 26
V. Group Mean Maze Transit Time Per Day in Seconds • .27
iv
CHAPTER I
INTRODUCTION
Researchers have found that the mental deficiency of
phenylketonuria in children can be prevented or favorably
modified if a special low-phenylalanine diet is started
early in life (26). Clinical findings have shown that this
restricted diet can be terminated at approximately age five
without any adverse effects even though the child continues
to have an abnormal metabolism (21, 26), However, recent
evidence of intrauterine retardation in offspring of phenyl-
ketonurias raises the question of whether or not the restrict-
ed diet should be re-established when a phenylketonuria mother
becomes pregnant (31, 32). The congenital retardation is
not the result of a genetic factor because the^ damaged chil-
dren in question are not genotypically phenylketonuria, It
is thought by Mabry that the abnormal maternal phenylalanine
metabolism has a direct in utero effect on the growing fetus (32)
In effect, this can be much worse than the child's inherit-
ing phenylketonuria because with phenylketonuria he can be
put on a therapeutic diet and the retardation arrested. It
has been found that with intrauterine retardation the damage
is inevitable and the child is born irreversibly retarded (^2),
This study was concerned with setting up a similar
situation wherein pregnant mice had an abnormally high
1
phenylalanine metabolism. Through physical and intellec-
tual assessment of their offspring, it would then be possible
to determine what effects the abnormal metabolism had during
pregnancy and whether or not a restricted diet need be re-
sumed at that time.
In order to better understand the present problems fac-
ing these treated phenylketonuria females, a review of the
history, dynamics, clinical course, and treatment of phenyl-
ketonuria is appropriate. In the early 1930's the Norwegian
physician and biochemist Ashborn Foiling was confronted with
two mentally retarded children who produced a strange odor (7),
Upon examining the children, Foiling discovered that the
urine of both reacted with ferric chloride to produce an
unusual green color. He was able to prove that this color
was due to the presence of phenylpyruvic acid, which he
crystallized in pure form from the urine sample. The strange
odor was found by Foiling to be linked with phenylacetate.
This same ferric chloride test and subsequent modifications
paved the way for present-day early detection and treatment
of phenylketonuria (7),
As news spread of the newly discovered "Polling's Dis-
ease," as it was called, screening programs were initiated
in institutions for the mentally retarded. These programs
resulted in incidence rates, genetic etiology, clinical
course, and eventual mapping out of the metabolic error (22).
The name phenylketonuria was suggested because phenyl-
pyruvic acid, the substance responsible for the green color
reaction with ferric chloride in the urine, is a phenylke-
tone (38)* The disorder has also been known as phenylpyru-
vic oligophrenia. Clinical usage, however, has allowed for
its rather lengthy name to be abbreviated simply as PKU.
This abbreviation will be used throughout the remainder of
this text.
Through screening programs it has been found that PKU
occurs in one-half to one percent of all institutionalized
mental defectives. From these results it has been estimated
that the disorder occurs once in every 20,000 to 40,000 live
births (22, 1), More extensive testing of blood phenylalanine
levels of new born babies in the United States implies that
the incidence approaches one in 10,000 live births (17),
Both sexes are equally affected, and all races tend to be
involved, although incidence is higher in Europeans (22)
and lower in Ashkenazi Jews (8) and Negroes.
It was early recognized that PKU was of, genetic origin
for the following reasons» It often involved more than one
child in the family1 the parents were normal, and approxi-
mately one in four children in the involved family had the
disorder (15). This information led to the conclusion that
PKU was inherited by a simple autosomal, recessive gene.
Thus, for each pregnancy of a couple heterozygous for the
PKU gene, there is a one in four chance that the child will
be PKU and a three in four chance of a phenotypically normal
child.
Langdellf a researcher in the area, referred to PKU as
a paradigm of the concept of a twisted mind from a twisted
molecule. He stated that
As a recessively inherited disease (not sex-linked), phenylketonuria occurs only if a baby inherits from each parent a "twisted" DNA (deoxynucleic acid) mole-cule that has one segment (gene) with disruption of the four amino acids that record the genetic code. Because of this defect, the DNA molecule produces an RNA (ribonucleic acid) that lacks the proper code message to form the enzyme, phenylalanine hydroxylase, that normally converts the essential amino acid phenylalanine to tyrosine in the liver. This chemical blockage dams up the normal metabolic flow, causing a rising tide of phenylalanine in the bloodstream that reaches abnor-mal levels 20 times the normal limit of 2 mg./100ml. A metabolite, phenypyruvic acid, "spills over" the kidney threshold into the urine after the serum pheny-lalanine level reaches 10 to 15 mg./lOO ml. (29, p. &9).
This "spilled over" phenylpyruvic acid is the same metabolite
that Foiling discovered in the urine samples of the two
mentally retarded Norwegian children, and it is the same
metabolite which led to the mapping out of the metabolic
error.
A point to be emphasized is that phenylalanine is an
essential amino acid. Amino acids make up proteins and
nearly all foods supply protein containing five per cent
phenylalaninej hence, a child who inherits PKU, although
normal at birth, will rapidly begin to build up a high pheny-
lalanine blood level due to the ingestion of milk, the basic
diet of all infants, which is high in protein.
Researchers believed that the continued high level of
phenylalanine or its related metabolites was responsible
directly or indirectly for the mental retardation. In 1953
dietary treatments low in phenylalanine were first described
(4, 45, 2). These diets have since proven to be adequate
in controlling the phenylalanine blood concentration and
result in a physically and mentally normal child (9, 21, 26).
Although the exact mechanism responsible for the ad-
verse effects of elevated levels of phenylalanine or abnormal
metabolites of phenylalanine metabolism has not been clari-
fied, a number of facts have emerged. Experimental evidence .
(18, 13, 35, 39) reveals an inhibitory effect on enzymen
systems active in significant metabolic and transport pro-
cesses. Serotonin, a most important neurohormone, and its
products are known to be inhibited in untreated PKU patients
and can be normalized under therapeutic dietary control (37).
In addition, there has been shown to be a failure or loss
of myelinatiort in about a third of PKU patients examined
histologically (11). On the other hand, there has been an
occasional case of untreated PKU reported without adverse
effects. This has served to point out the many yet unanswered
questions concerning the biochemical dynamic of this abnor-
mality.
The untreated PKU patient is born apparently normalj
then he begins to show retardation early in life, A subtle
change may be noticed at about three or four months of age•
Apathy is evident and development is slowed down or arrest-
ed until by two or three years of age most are in the severe-
ly retarded range of intelligence. Ninety per cent of the
known untreated cases have fallen into this bracket (22, 36).
Irritable behavior, convulsion seizures, and abnormal elec-
troencephalograms have been frequent. Other signs have in-
cluded a positive Babinski, ankle clonus, eczema, the musty
odor of phenylacetate, and the characteristic pale skin,
bleached hair, and blue eyes resulting from disturbed tyro-
sine metabolism that interferes with the formation of melanin
pigmentation.
For untreated PKU children the average age for sitting
alone is from twelve to fifteen months 1 the average age for
walking is two and one-half years, for talking three and one-
half years. Some never learn to walk, and many never learn
to talk.
Two basic tests are used for screening infants, urine
tests and blood tests. Urine tests include the Ferric Chlor-
ide Test Tube Test (15)» Ferric Chloride Diaper Test (6),
Phenistic (R) Test (40), and the Dinitrophenylhydrazine
Test Tube Test (39)* The Ferric Chloride Test Tube Test is
the oldest, best known, and tnost widely used of any of the urine
tests for PKU. The Dinitrophenylhydrazine Test Tube Test
is the most sensitive and reliable of the urine tests but
is prohibitively expensive and difficult. The Guthrie
Inhibition Assay Test is the major blood test used (16). It
is a simple and inexpensive test and can be performed in
large volume. Blood tests are preferred over urine tests
for one major reason. For phenylpyruvic acid to be present
in the urine and result in a positive ferric chloride test,
the phenylalanine blood level must exceed renal threshold.
By the time this level is attained, neural damage can already
have taken place. On the other hand, the blood test can
detect an abnormal phenylalanine level in the blood before
it builds up to renal threshold and is capable of beginning
neural damage.
If screening tests show positive three consecutive times,
then quantitative serum phenylalanine determinations are used
for final confirmation and clinical care (16). These various
methods (3, 19, 20, 28, 33) are too complex for description
here.
With final confirmation of PKU it is recommended that
the child be put on the therapeutic diet as early as possible,
because the greatest damage tends to take place in the first
months of life (32). Knox (26) has calculated that an aver-
age of half an I.Q. point is lost each week that treatment
is delayed within the first three years of life. Older pa-
tients have to be considered individually, but it has been
found that the diet can lead to limited intellectual, emotion-
al, and physical improvement even in these progressed cases (10).
8
All low-phenylalanine diets are based on synthetic
foods which provide all the essential amino acids excluding
phenylalanine. This material is commercially produced by
hydrolysing milk casein and removing phenylalanine by fil-
tration. Other amino acids removed in the processing must
be replaced. It is then fortified through activated char-
coal.
The aim of this diet is to lower the blood phenylalanine
from the abnormally high levels caused by the disease to
near-normal levels while promoting normal body growth by
providing the essentials for good nutrition. However, this
diet is so low in phenylalanine that it must be supplemented
with carbohydrates, oil, minerals, and vitamins. Otherwise,
i fiere would be poor growth and a paradoxical rise in serum
phenylalanine due to catabolism of body protein.
Low-phenylalanine products available in Europe include
Cymogren (12) and Minafen (34). In the United States Lo-
fenalac (30) has replaced Ketonil (25). Dietary tables ac-
companying these products enable maintenance of near-normal
phenylalanine levels. In addition, repeated blood tests are
needed to guide adjustment of this diet.
Presently, scattered experiences have indicated that
children over three have maintained their I.Q.'s upon termin-
ation of the diet. Although the majority of researchers
feel that age four is a safe time for discontinuing the diet,
some believe that termination should not occur until adoles-
cence (27, 5).
In addition, recent evidence (31) suggests that treated
PKU females who are desirous of pregnancy may have to return
to their low-phenylalanine diet during the entire pregnancy.
This particular idea is beginning to receive added attention
and is felt to have been associated with many unexplained
cases of mental retardation which might have been prevented.
In support of this concept Mabry (32, p. 5) reported
the incidence of three PKU mothers giving birth to children
who were not genetically PKU, but who were, nevertheless,
mentally retarded. He felt that "this observation supports
the concept that a high blood phenylalanine level.in the preg-
nant woman causes irreparable damage to the otherwise normal
brain of her unborn child."
Stevenson (42) gave an account of two PKU sisters on
non-restricted diets whose pregnancies had resulted in six-
teen spontaneous abortions, six offspring who died in in-
fancy, and four presently surviving children. He stated that
3-11 live births to these females had varying combinations
of premature birth weights, microcephaly, cardiac defect,
mental and physical retardation, dislocated hips#and stra-
bismus. He emphasized that none of these children had PKU,
^ Numerous observations (14, 21, 24, 26) have shown that
before birth and the early months of life are the most vul-
nerable period for the brain. These observations aid in
understanding why the PKU child*s brain is not damaged when
he is taken off the therapeutic diet at approximately age
10
five. That is to say that even though the child's phenylala-
nine blood concentration returns to its previously harmful
levels upon termination of the diet, there is no neurologi-
cal damage evidenced because the brain has passed its vul-
nerable growth period,
Kerr and his colleagues (2*0 have recently demonstrated
that amino acids are actively transported across the mammal-
ian placenta. In addition, he found that the concentrations
in fetal blood are higher than the maternal concentrations.
Kang and Paine (23) found that pregnant; women heterozy-
gous for PKU had a higher blood phenylalanine concentration
than both normal pregnant and non-pregnant PKU heterozygote
controls. Mabry (32) felt this evidence enough to suggest
the possibility that the normal placental process might mag-
nify the maternal biochemical error and produce an even more
profound disturbance in the fetus.
Mabry emphasized that all these observations supported
the concept of transplacental hyperphenylalaninemia-induced
brain damage. He stressed this by statingj
When family studies, experimental data and gene-tic information described above are considered as a whole, we are led to believe.that the phenyketonuric mother's hyperphenylalanmemia and related plasma alter-ations damage the brain of her otherwise normal fetus and that maternal phenylketonuria is causing a clinical-ly significant number of retarded children. An immedi-ate implication is that this form of mental retardation is avoidable, either by prevention of the pregnancy or by induction of a lowered blood phenylalanine con-centration during gestation (32, p. 1336) ,
11
Although a number of researchers (41, 44) have simulated
PKU in animals through dietary manipulation and injection of
phenylalanine, nearly all of this was induced after birth.
There has been little work in the area of simulating a situ-
ation wherein intrauterine effects could be studied.
The one related study, to date, was performed by Thomp-
son and Kano (43). Through dietary manipulation a hyper-
phenylalaninemia situation was induced in pregnant female
rats, and offspring were evaluated. No significant physical
or intellectual deficits were observed; however, the authors
noted a temperament or emotional difference between the con-
trol and experimental groups. They concluded that there was
an emotional change but not an intellectual one. •
Considering the lack of research in this area and its
importance in the field of mental retardation and learning,
it was the purpose of this study to simulate a situation of
PKU in pregnant mice in order to study the physical and in-
tellectual difference between offspring of pregnant mice
intubated with a solution of phenylalanine and offspring
of pregnant mice intubated with a placebo. Fetal and post-
partum deaths were utilized as physical measures while learn-
ing ability in a maze was used as an intellectual measure.
12
The assumption was made that a high maternal phenylala-
nine blood concentration in mice would lead to significantly
more physica-l and intellectual abnormalities than in the off-
spring of mice having a normal maternal phenylalanine blood
concentration. Having made the assumptions that phenylala-r
nine is in a solution, that such a solution can cross the
placental barrier to cause intrauterine growth retardation,
and that the earlier in pregnancy that the transport takes
place, the greater the retardation effect, the following
hypotheses were assumed.
1. Offspring of pregnant mice intubated with a pheny-
lalanine solution will show a signigicantly higher incidence
of morbidity and/or mortality than those offspring of preg-
nant mice intubated with a placebo.
2. Offspring of pregnant mice intubated with a pheny-
lalanine solution will perform significantly less adequately
in a learning situation than will offspring of pregnant mice
intubated with a placebo.
3. Offspring of pregnant mice intubated with a pheny-
lalanine solution early in pregnancy will show a signifi-
cantly greater physical and intellectual deficit than off-
spring of pregnant mice intubated with a phenylalanine so-
lution late in pregnancy.
CHAPTER BIBLIOGRAPHY
1. Armstrong, M, D. and N. L, Low, "Phenylketonuria VIII. Relation Between Age, Serum Phenylalanine Level, and Phenylpuruvic Acid Excretion," Proceedings of the So-ciety for Experimental Biology and Medicine, XCIV (January, 1957). I W - l C
2. Armstrong, M. E. and F. H. Tyler, "Studies on Phenyl-ketonuria I, Restricted Phenylalanine Intake in Phenylketonuria," Journal of Clinical Investment, XXXIV (March, 1955), 565-580.
3. Berry, H. K., "Paper Chromotographic Method for Estima-tion of Phenylalanine," Proceedings of the Society for Experimental Biology and MedicineT XCV (May. 1957). 71-73.
4. Bickel, H,, J. Gerrard, and E. M. Hickmans, "Influence of Phenylalanine Intake on Phenylketonuria," Lancet. II, (October 17, 1953), 812-813.
5# Bickel, H., Chapter in Phenylketonuria, edited by Frank Lyman and Charles C. Thomas, Springfield, 111., 1963.
6. Centerwall, W, R,, R. F. Chinnock, and A, Pusavat, "Phenyl-ketonuria! Screening Programs and Testing Methods," American Journal of Public Health. L (November, i960), 1667-1677.
7. Centerwall, W. R. and S. A. Centerwall, "Phenylketonuria (Foiling*s Disease)! The Story of its Discovery," Journal of the History of Medicine and Allied Sciences. XVI (July, 1951), 292-295.
8. Centerwall, W. R. and C. A. Neff, "A Case Report of Children of Jewish Ancestry," Archives of Pediatrics. LXXVIII (October, 1961), 3 7 9 - 3 ^
9. Centerwall, W. R., S. A. Centerwall, V. Armon, and L. B. Mann, "Phenylketonuria II1 Results of Treatment of Infants and Young Children, A Report of Ten Cases," Journal of Pediatrics. LIX (July, 1961), 102-118.
10. Clader, D• E,, "Accelerated Intellectual Growth and Per sonality Development as Seen in Phenylketonuria Sub-jects during Medical Treatment," American Journal of Mental Deficiency. LXII (November, 1957), 538-5^2.
1 1
14
11. Crome, L, and C. M, B. Pare, "Phenylketonuria! A Re-view and Report of the Pathological Findings in it-Cases,M Journal of Mental Science. GVI (April, i 9 6 0 ) , 8 6 2 - 8 6 3 ,
12. CymogramR. Allen and Hanbury's Ltd., Bethnal Green, London, E. 1 , England.
13. Davison, A. N. and M. Sandler, "Inhibition of 5-Hydroxy-tryptophan Decarboxylase by Phenlalanine Metabolites," Nature, (London), CLXXXI (January 18, 1958), 186-187.
14. Davison, A, N. and J. Dobbins, "Myelination as a Vulner-able Period in Brain Devilopment," British Medical Bulletin. XXII (January, 1 9 6 6 ) , 4o -W7^
15. Foiling, A., "Phenylpyruvic Acid as a Metabolic Anomaly in Connection with Imbecility," Nord. Med. TIDSKR.. XIII (March, 193*0. 1054-1059. "
16. Guthrie, R. and A. Susi, "A Simple Phenylalanine Method in Large Populations of Newborn Infants," Pediatrics. XXXII (September, 1 9 6 3 ) , 3 3 8 - 3 4 3 . 1
17. Guthrie, R. and S. Whitney, "Phenylketonuria, Detection in the Newborn Infant as a Routine Hospital Procedure," Children's Bureau Publication. No. 4.19, U. S. De-partment of Health, Education, and Welfare, Washing-toft, D. C., 20201, 1964,
18. Hanson, A., "Inhibition of Brain Glutamic Acid Decar-boxylase by Phenylalanine Metabolites," Naturwis-senschaft , VL (October, 1958), 423.
19» Henry, R . J . , C , Sobel, and N. Chiamori, "Method for Determination of Serum Phenylalanine with Use of the Kapeller-Adler Reaction," American Medical Associ-ation Journal of Children. VIC 60^-608•
20, J, B., G. K. Summer, M. W. Pender, and Norris 0» Roszel, "An Automated Procedure for Blood Phenyla-lanine," Clinical Chemistry. XI (May, 1 9 6 5 ) , 541-546.
21. Horner, F. A. and C. W. Streamer, "Effect of a Pheny-lalanine—Restricted Diet on Patients with Phenyl-ketonuria! Clinical Observations in Three Cases," Journal of the American Medical Association, CLXT (August, 1956T 1628-1630"
15
22. Jervis, G. A., "Phenylpyruvic Oligophrenia! Introduc-tory Study of 50 Cases of Mental Deficiency Asso-ciated with Excretion of Phenylpyruvic Acid," Archives of Neurology and Psychiatry. XXXVIII (November, 1937), 944-963.
2 3 . Kang, E. and R, S. Paine, "Elevation of Plasma Pheny-lalanine Levels During Pregnancies of Women Hetero-zygous for Phenylketonuria," Journal of Pediatrics. LXIII (August, 1963),283-289.
24. Kerr, G. R. and H, A, Waisman, "Phenylalanine! Trans-placental Concentrations in Rhesus Monkeys," Science, CLI (May, 1 9 6 5 ) , 6 8 5 - 6 9 5 .
R 25. Ketonil . Merck, Sharp and Dohme, Philadelphia, Pa.
26. Knox, W. E., "An Evaluation of the Treatment of Phenyl-ketonuria with Diets Low in Phenylalanine," Pediatrics. XXVI (July, I960), 1-11.
27. Koch, R., K. Fishier, S. Schild, and N. Ragsdale, "Clini-cal Aspects of Phenylketonuria," Mental Retardation. II (February, 1964), 47-5*1-.
28. LaDu, B. N. and P. J, Michael, "An Enzymatic Spectro-photometry Method for the Determination of Pheny-lalanine in Blood," Journal of Laboratory and Clini-cal Medicine. LV (March, I 9 6 0 T , 491-496.
29. Langdell, John I., "Phenylketonuria! Childhood and Adolescence," Current Psychiatric Therapies. V (Annual Publication, 1965), 49-56.
R " 30. Lofenalac . Mead Johnson and Company, Evansville, Ind.
31. Mabry, C. C., J, C. Denniston, T. L. Nelson, and C. D. Son, "Maternal Phenylketonuria! A Cause of Mental ' Retardation in Children Without the Metabolic Defect," New England Journal of Medicine. CCLXIX (December, 1963). 1404-1408.
32. Mabry, C. C., J. C. Denniston, and J. G„ Caldwell, Mental Retardation in Children of Phenylketonuria Mothers," New England J ournal of Medicine. CCLXXV (December, 1 9 6 6 ) , 1331-1336.
33• McCaman, M. W. and E. Robins, "Fluorimetric Method for the Determination of Phenylalanine in Serum," Jour-
j u r a t o r y and Clinical Medicine. LIX (MayT"
16
R 34. Mjnafen » Trufood Ltd., 113 Newington Causeway, London,
S. E. I., England.
35. Neame, K, D,, "Phenylalanine as Inhibitor of Transpost of Amino Acid in Brain," Nature (London), CIIIVC (October, 1961), 173-174.
36 . Paine, R, S. "The Variability in Manifestations of Untreated Patients with Phenylketonuria (Phenylpy-ruvic Aciduria)," Pediatrics, XX (August, 1957)» 290-302.
37• Pare, C. M. B., M. Sandler, and R. S, Stacey, "Decreased 5-Hydroxytryptophan Decarboxylase Activity in Phenyl-ketonuria," Lancet, II (November, 1958), 1099-H01.
38 . Penrose, L. S,, "Inheritance of Phenylpyruvic Amentia (Phenylketonuria)," Lancet, II (July, 1935), 192-19*f.
39# Penrose, L. A. and J, H. Quastel, "Metabolic Studies On Phenylketonuria," Biochemical Journal. XXXI (February, 1937)» 266-274.
R 40. Phenistix . Ames Company, Inc., Elkhart, Ind.
41. Polidora? V. J., D.E. Boggs, and H. A. Waisman, "A Behavioral Deficit Associated with Phenylketonuria in Rats," Proceedings of the Society for Experimental Biology and Medicine. CXIII (September, 1 9 6 3 ) , 817-820.
42. Stevenson, R. E. and C. C. Huntley, "Congenital Mal-formations in Offspring of Phenylketonuria Mothers," Pediatrics. XL (July, 1967), 33-45,
43. Thompson, W. R. and K. Kano, "Effects on Rat Offspring of Maternal Phenylalanine Diet During Pregnancy," Journal of Psychiatric Research. Ill (Mav. I960. 91-98.
4^. Waisman, H. A. and H. P. Harlow, "Experimental Phenyl-ketonuria in Infant Monkeys," Science. CIIIL (Febru-ary, 1965)1 6 8 5 - 6 9 5 .
45. Woolf, L. I., R. Griffiths, and A. Moncrieff, "Treat-ment of Phenylketonuria with a Diet Low in Pheny-lalanine," British Medical Journal. I (January, 1955)» 56-64.
CHAPTER II
METHOD
Subjects
Breeding stock consisted of sixty naive mice of the
colony C 57 BI/6J from the Jackson Memorial Laboratories.
All mice were four months old at breeding. The resulting
offspring, which made up the experimental and control groups#
numbered one hundred and forty. These were tested at six
months of age. This particular strain was selected because
it has been found to be generally resistant to the effects
of phenylalanine and therefore does not bias the results in t
studies utilizing this amino acid. All subjects were main-
tained on ad libitum food and water.
Apparatus
A four-unit, water-filled, T maze was employed. The
maze was similar in design to one used by Biel (1) for rats
but was scaled down to accommodate mice (see Appendix).
Construction of the maze was designed to conform to the
dimensions of an unpainted stainless steel tank, 18" long,
14" wide, and 14" deep. The 14"-high walls of the maze proper
were constructed of eighteen—gauge galvanized sheet metal and
were left unpainted. The goal consisted of an 8"-long wire
ramp which ascended at a 45° angle to a wire platform 2" wide
18
Water depth was 7", preventing subjects from touching
the bottom, and the walls were slippery enough to prevent
resting. Water temperature was held at a constant 15 centi-
grade as a means of motivation. Also held constant were room
temperature, lighting and sound. A standard stopwatch was
used to time runs.
Procedure
Breeding was commenced by placing two females with one
male per cage. Daily pregnancy tests were made at 9:00 A.M.
This was found by a pilot study to be the optimal time for
detection of vaginal or coital plugs. The vaginal plug method,
one of the most reliable, according to Rugh (2), consists of
observing for the occurrence of dried mucous formation at the
opening of the vagina, in addition to local swelling and red-
dening. The plug must be sought within a few hours of forma-
tion because it soon dried out and crumbles and'can-be torn
down by urination.
Upon detection of pregnancy, each female was randomly
assigned to an Early, Middle or Late Pregnancy Experimental
or an Early, Middle or Late Pregnancy Control group and
placed in an individual cage to await daily intubation. Sub-
jects remained in these individual cages until the pregnancy
came to term.
At partruition the offspring were counted and observed
for morbidity, then left with their mothers until weaning at
19
•three weeks of age. All efforts were exercised to avoid
excessive handling of the pups since this could have led
to their destruction by the mother.
At four weeks of age the pups were isolated to prevent
mating. Males were caged individually, and females were i)
placed two to a cage.
All subjects were checked daily for physical progress
and activity level until maze testing began at six months
of age. A pilot study showed this age to possess sufficient
strength for the swimming task.
The first day of maze activity began with each mouse
receiving three training trials in the 18" straight channel.
This consisted of placing the mouse at the end of the straight
channel opposite the exit ramp and timing him to assess swim-
ming ability. An additional purpose was to familiarize the
subjects with swimming in a straight channel. A guillotine
door closed off the remaining channels.
Actual maze transit was begun on the second day and
continued for a total of eight days, A maze transit run
consisted of placing an individual subject in the end of the
start channel with his head facing in the correct direction.
Timing began the moment he touched the water and was stopped
the moment his forepaws touched the exit ramp. Each mouse
was allowed three minutes to reach the goal. In the event
the subject could not complete the maze in three minutes,
he was guided through to the goal with a glass pestla As
20
soon as he reached the ramp and climbed to the platform, each
subject was dried with a clean laboratory towel and placed in
his individual cage. All subjects received three trials per
day for eight days. With each trial, maze transit time and
number of errors were recorded. An error was the entry of
the complete body into a cul. Throughout testing only one
mouse at a time was allowed in the maze.
Intubations
Intubations were performed with a one-cubic-centimeter
tuberculin syringe fitted with an 18-gauge needle. Fastened
over the tip of the needle was a three-inch section of #190
Intramedic Polyethyline Tubing.
A table-mounted wire triangle with a one-inch base and
five-inch sides was employed to assist in keeping the mouse's
mouth open as the tube was inserted into its esophagus. The
phenylalanine was then introduced into the stomach for
absorption, by depressing the plunger in the syringe.
All intubation dosages of the experimental amino acids,
DL phenylalanine, were made according to the ratio suggested
by Schalosk and Klopfer (3). This suggested ratio of three
grams DL phenylalanine per kilogram of body weight resulted
in an intubation dosage of .18 milliliter of phenylalanine
solution. The phenylalanine solution was prepared as follows!
five grams of DL phenylalanine were added to 100 milliliters
of sterile physiological saline (isotonic sodium chloride
21
the solutions were clear. Intubations were administered
at 40° centigrade,
A standard intubation procedure was carried out for
all subjects^ in that all experimental subjects received .18
milliliters of DL phenylalanine solution and all control
subjects received ,18 milliliters of sterile physiological
saline. Extreme caution was exercised in seeing that all
subjects were handled in the same way throughout intubation
and that the only treatment differential was what was being
intubated and not how, or the amount.
The intubation schedule according to groups was as
follows»
The Early pregnancy groups (both experimental and con-
trol) received their first intubation on the seventh day of
pregnancy. These constituted the 1?E and 1?C groups respec-
tively, They then received one intubation per day every day
thereafter until partuition.
The Middle pregnancy groups (both experimental and con-
trol) received their first intubation on the twelfth day
of pregnancy. These constituted the 12E and 12C groups,
respectively. They then received one intubation per day
every day thereafter until parturition.
The Late pregnancy groups (both experimental and con-
trol) received their first intubation on the seventeenth
day of pregnancy. These constituted the 7E and ?C groups,
22
respectively. They then received one intubation per day
every day thereafter until partuition.
Attempt was made to hold the variables of time, tempera-
ture, place, and sound constant throughout intubations.
CHAPTER BIBLIOGRAPHY
1. Biel, William C,, "Early Age Differences in Maze Per-formance in the Albino Rat," Journal of Genetic Psy-chology . LVI (June, 19^0), 439-^53.
2. Rugh, Roberts, Laboratory Manual of Vertebrate Embry-ology, Minneapolis, Minn., Burgess Publishing Company, 19^1-1961.
3. Schalock, R. L. and F. D. Klopfer, "Phenylketonuria! Enduring Behavioral Deficits in Phenylketonuria Rats," Science. CLV (February, 196?)» 1033-1035*
23
CHAPTER III
RESULTS
In an attempt to measure the effects of certain mater-
nal amino acid levels on the developing mouse fetus, thirty-
six female mice were administered DL phenylalanine at various
stages of pregnancy.
Table I represents the mortality rates of all offspring
of the thirty-six subjects as observed from birth to ten days
following birth.
TABLE I
TOTAL PERCENT OF SURVIVING OFFSPRING IN EACH GROUP
Group 17C 17E 12C 12E 7C 7E
93% 100% 100% 50% 97% 27%
The similarity of all control groups indicates no sig-
nificant effects owing to intubation of a placebo. In addi-
tion, the similarity of the Late Pregnancy Experimental
Group (17E) to its matched Control Group (17C) indicates no
significant effects owing to the experimental treatment. How-
ever, it is clearly shown that the Middle Pregnancy Experi-
mental Group (12E) and Early Pregnancy Experimental Group (7E)
experienced a treatment effect.
24
25
A t test for differences between proportions was used
to determine the significance of differences between the con-
trol and experimental groups at each level. Table II con-
tains the results of these tests,
TABLE II
PERCENTAGES AND LEVELS OF SIGNIFICANCE BETWEEN C AND E GROUPS
Groups Percent Survivors N t df P
17C 17E
93 100
28 30
1.36 56 P=.l
12C 12E
I
O
O O
30 15 ^3 P=,001
7C 7E
97 27
29 8 5.51 35 P=.001
It can be seen in Table II that no significant differ-
ence was found between 1?E and 1?C groups. On the other
hand, a high degree of significance is found between both
12E and 12C and between 7iS and 7C, thtis indicating a treat-
ment differential for both these groups.
In addition, t tests for proportions were calculated
between all E groups. Table III contains the results of
these tests.
The high degree of significance illustrated in Table III
between 17E and 12E and between 17E and 7E supports a relation
TABLE III
PERCENTAGES AND LEVELS OF SIGNIFICANCE BETWEEN E AND E GROUPS
26
Groups Percent Surviors N t df P
1?E 12E
100 50
30 15
4,4-2 ^3 P=,001
17E 100 30 5.90 36 P=,001
12E 7E
50 27 C
OU
l O
CO
• 21 •
II ! i
between the stages of pregnancy in which phenylalanine intu-
bation took place and the resulting rate of mortality. How-
ever, no such significant difference was found to exist
between 12E and 7E as concerning this variable.
Table IV contains the results of eight days of maze
training and testing as expressed in mean errors for each
group per day,
TABLE IV
GROUP MEAN MAZE ERRORS PER DAY
Group 17C 17E 12C 12E 7C 7E
Day 1 2 3 1.86 2.80 2.30 4 Day 2
o
00 • H 1.78 1.36 1.80 1.70 1.75
Day 3 .97 1.15 1.36 .88 1.30 1.70
Day 4 .82 .78 .83 .38 1.75 1.12
Day 5 .50 .65 .33 .70 .87 .68
TABLE IV—Continued
27
Group 17C 17E 12C 12E 7C 7E
Day 6 M . 40 . 8 3 .87 • 68
Day ? . 3 3 . 2 3 .07 . 39 • 54 . 58
Day 8 . 4 0 . 1 4 . 1 0 . 3 3 .12 .08
Due to the lack of variability illustrated in Table IV,
no further statistics were computed for maze errors. It was
then accepted that no intellectual deficit was exhibited in
the form of maze error.
Table V represents the mean maze transit time for each
TABLE V
GROUP MEAN MAZE TRANSIT TIME PER DAY IN SECONDS
Group 17C 17E 12C 12E 7C 7E
Day 1 63.5 72 40 38 48 6 6 . 8 Day 2 46 3 5 . 6 5 3 . 5 35 28 25 Day 3 44 26 • 6 36 35 2 1 . 7 13 Day 4 33 2 1 , 6 36 31 30 1 2 . 3 Day 5 3 2 . 5 28 3 3 . 6 4 0 , 7 2 5 . 7 2 2 . 6 Day 6 2 7 . 4 1 7 . 3 23 30 38 10.5 Day 7 3 4 . 5 1 6 . 6 28.5 4 2 . 6 5 2 . 7 20.6 Day 8 21 1 1 . 6 1 3 . 9 3 7 . 9 4 9 . 2 1 4
group per day. The mean for the three trials per day is
expressed in seconds.
28
Again, owing to the erratic spread of variability, no
further statistics were computed for maze transit time as
a measure of intelligence. It was then accepted that there
was no intellectual deficit owing to experimental treatment
as measured by maze transit time.
/ /
CHAPTER IV
DISCUSSION
The results in Table II revealed that there was a sig-
nificant difference between the two earliest experimental
groups and their respective control groups. The significant
differences illustrated that the conditions of intubation
of phenylalanine and intubation of a placebo were signifi-
cantly different from each other at a level greater than
.001 for the 12E and 12C and 7E and 70 pregnancy groups.
However* no significant difference was found to exist between
the 7E group and the ?C group*.
This significant difference between the 12E and 12C
and 7E and 7C pregnancy groups indicated that an increased
mortality rate could result due to a high maternal level of
phenylalanine during the middle and early stages of preg-
nancy. Although the levels of significance were the same,
it can be noted in Table II that the percentage of mortal-
ity was approximately twice as great with each earlier level
of pregnancy. This indicates that the earlier in pregnancy
the intubation took place, the greater the rate of mortality
in offspring.
The results in Table III showed a significant difference
between the ?E group and 12E group, and between the 17E group
29
30
and the ?E group. However, no significant difference was
found to exist between the 12E group and ?E group. This
lack of significance is felt to arise from the low number
of subjects in each of these seven-and twelve-day experiment-
al groups due to the higher mortality rates experienced.
However, the significance of the other experimental groups
warrants acceptance thattthe earlier in pregnancy the intu-
bation takes place, the greater the mortality rate.
This evidence lends some support to the theory that
elevated maternal levels of phenylalanine during pregnancy
can lead to an increased mortality rate in offspring^ and
in turn confirms the hypothesis that offspring intubated
with a phenylalanine solution will show a significantly higher
incidence of morbidity and/or mortality than those offspring
intubated with a placebo.
This evidence also partially confirms the additional
hypothesis that offspring of pregnant mice intubated with a
phenylalanine solution early in pregnancy will show a sig-
nificantly greater physical (mortality) and intellectual
deficit than offspring of pregnant mice intubated with a
phenylalanine solution late in pregnancy. Byypartial con-
firmation is meant that the physical deficit was confirmed
but that the intellectual deficit was not. This lack of
intellectual deficit will be discussed later.
On the basis of the assumption that phenylalanine is
in a solution and of relatively simple molecular structure
31
and that such a solution can across the placental barrier to
cause intrauterine damage to the developing fetus, the re-*
suits of this study are compatible with the observations of
Stevenson ( $) who gave an account of two PKU sisters on
non-restricted diets whose pregnancies had resulted in six-
teen spontaneous abortions, six offspring who died in infancy
and four surviving children. He stressed that all surviving
children had varying combinations of physical and mental
retardation and that none of these children were genetically
PKU, This evidence would definitely indicate intrauterine
retardation.
Although the results of maze testing shown in Tables
IV and V indicated no intellectual damage, it is felt that
the remaining offspring in the earlier groups should be clas-
sified as a survival of the fit. That is, when an animal
experiences prenatal damage or is in some way defective,
it is generally reabsorbed or dies soon after birth. Hence,
it is felt that if the aborted animals had survived, they
would have shown an intellectual deficit and that the surviv-
ing animals were stronger and less susceptible to being damaged
by the adverse intrauterine environment. In support of this
concept is the observation that the mice in the earliest
experimental group were faster and more hyperactive than
any of the others. Nevertheless, the evidence of increased
fetal and congenital mortality, as revealed in the two earli-
er experimental groups, lends much support to the various
32
theories which suggest adverse fetal effect due to increased
maternal phenylalanine levels during pregnancy.
A leading researcher in the area, Mabry (2), emphasized
that the treated PKU mother's hyperphenylalaninemia and re-
lated plasma alterations were causing a significant amount
of intrauterine brain damage and consequent mental retarda-
tion. He stated that "this form of mental retardation is
avoidable, either by prevention of the pregnancy or by
induction of a lowered blood phenylalanine concentration
during gestation."
Again, although no intellectual deficit was observed
in this study, it is felt that the increased mortality rate
is sufficient evidence to warrant support of this same the-
ory of hyperphenylalaninemia-induced brain damage.
What happens in hyperphenylalaninemia, in essence, is
that the PKU mother's phenylalanine blood concentration is
at a dangerously high level. This level is not dangerous to
the mother because her brain has already passed the vulner-
able stages of development at approximately age fivet How-
ever, since Kerr (1) has demonstrated that amino acids are
actively transported across the mammalian placenta, it is
feasible to assume that the elevated maternal levels of pheny-
lalanine would have a definite effect on the developing
fetus. In addition, Kerr (1) found that phenylalanine con-
centrations in fetal blood were higher than those in maternal
bloodi therefore, this magnified effect would almost assure
33
fetal damage. Since this elevated phenylalanine level has
been found to be damaging, this situation is actually much
worse than a child's merely inheriting PKU, because with PKU
the child can be placed on a low-phenylalanine diet and pre-
vent brain damage, whereas in the case of hyperphenylalani-
nemia-induced brain damage, the child is irreversibly re-
tarded before birth.
A solution, as suggested by Mabry (2), would be to
return the mother in question to the low-phenylalanine diet
immediately upon confirmation of pregnancy. Actually, con-
sidering that the greatest amount of damage (in this study)
took place early in pregnancy, it would be advisable for
these PKU mothers to plan each child and to make attempts to
resume the low-phenylalanine diet before conception, thereby
avoiding the profound damage which could occur in the first
trimester. Likewise, since little or no damage occurred in
the late groups (in this study), then resumption of a normal
diet before partruition could be considered.
However, more research is needed in this area before
any definite conclusions can be drawn. The limitations of
this study must also be taken into consideration.
To begin with, the subjects used in this study were
mice, as opposed to humans, thus presenting problems of
extra-polation over such a wide range of the phylo-genetic
scale. Secondly, although the dosage level was validated in a
3^
it had on each individual mouse; that is, it was not precise-
ly known how much each individual mouse's phenylalanine blood
level was increased, but it could only be assumed that each
subject experienced the same effect from having received the
same dose,
In the one related study to date, Thompson and Kano (3)
found no intellectual or physical damage, but they did con-
clude that there was a tempermental or emotional change.
Their study differed from this one in that they used rats
rather than mice, and diet was used rather than intubation.
Also the fact that they employed urine tests to assess pheny-
lalanine blood concentrations might lend more validity to
their study. However, their phenylalanine dosage level as
expressed in relation to kilogram body weight was not statedi
hence it i's assumed that the difference in dosage levels
was an influencing factor in the conflicting results of
these two studies.
The intubation method of administering phenylalanine
solutions was selected because it presented less danger of
puncturing vital organs; as compared to interperitoneal in-
jection, and there was also less danger of aseptic needle
conditions, as compared to the injection method. On the
other hand, there was a danger that the subjects would re-
gurgitate the solution, but this occured only a few times
in the course of hundreds of intubations.
35
Another variable to be considered is age of offspring
at testing. It might be argued that the testing should have
occured before six months of age. Taking this into considera-
tion, a pilot study was conducted, and it was found that the
subjects did not possess sufficient strength to perfrom the
swimming task before five to six months of age.
Another suggested variable concerned the effects of
the high maternal phenylalanine bloo(| concentration on lac-
tation, which if reduced could lead to a high infant mor-
tality rate. Again, a pilot study was conducted in which
three litters of 7E pups were nursed by three 7C mothers,
and the three 70 pups were nursed by three ?E mothers. This
resulted in a seventy per; cent mortality rate among the ?E
offspring (who were nursed by a normal mother), and only one
death among the 70 offspring (who were nursed by a high pheny-
lalanine mother). It was concluded that there was no adverse
lactation effect due to a high maternal phenylalanine con-
centration.
On the basis of the experimental evidence and the assump-
tions that were made, it was concluded that a high maternal
phenylalanine blood concentration in mice,would result in a
high rate of fetal and congenital mortality in offspring,
and that the earlier in pregnancy that this phenylalanine
blood concentration was elevated, the more profound the effect.
However, ittwas not concluded that a high maternal phenyla-
lanine blood concentration would result in an intellectual
36
deficit in the offspring, as hypothesized. It is to be recog-
nized that these conclusions are justified only if the limi-
tations of this study are taken into consideration#
CHAPTER BIBLIOGRAPHY
1, Kerr, G, R. and H. A, Waisman, "Phenylalaninet Trans-placental Concentrations in Rhesus Monkeys," Science« CLI (May, 1965). 685-695.
2, Mabry, C. C,, J. C. Denniston, T. L, Nelson, and C. D, Son, "Maternal Phenylketonuria} A Cause of Mental Retardation in Children Without the Metabolic Defect," New England Journal of Medicine. CCLXIX (December, T9S3), 140^-1^08.
3, Stevenson, R, E. and C, C, Huntley, "Congenital Malfor-mations in Offspring of Phenylketonuria Mothers," Pediatrics. XL (July, 1967)» 33-^5.
CHAPTER V
SUMMARY AND RECOMMENDATIONS
Summary
Two hundred black, naive mice were used as subjects in
the present study. Sixty subjects were mated and the re-
sulting thirty-six pregnant females were matched at three
different levels of pregnancy in a high maternal phenylala-
nine blood concentration-normal maternal phenylalanine blood
concentration relationship, and were intubated with either
DL phenylalanine or placebo. The offspring were physically
and intellectually assessed in an attempt to measure the ef-
fects of a high maternal phenylalanine blood concentration
on offspring. This study also attempted to test the theory
that the state of hyperphenylalaninemia in PKU mothers is
causing a clinically significant number of retarded chil-
dren which could be prevented.
The following hypotheses were testedi
1. Offspring of pregnant mice intubated with a pheny-
lalanine solution will show a significantly higher incidence
of morbidity and/or mortality than those offspring of preg-
nant mice intubated with a placebo.
2. Offspring of pregnant mice intubated with a pheny-
lalanine solution will perform significantly less adequately
38
39
in a learning situation than will offspring of pregnant mice
intubated with a placebo.
c. Offspring of pregnant mice intubated with a pheny-
lalanine solution early in pregnancy will show a significant-
ly greater physical and intellectual deficit than offspring
of pregnant mice intubated with a phenylalanine solution
late in pregnancy.
Significant differences were found to exist between
some but not all treatment conditions. Statistical analy-
sis of the data revealed a physical deficit which increased
with intubations that took place in the early stages of preg-
nancy, but the analysis did not support an intellectual dif-
ference.
These results indicated that the high maternal pheny-
lalanine blood concentration during pregnancy can lead to a
high mortality rate in offspring arid that the earlier in
pregnancy that this high level exists,, the more profound the
damage.
Recommendations
On the basis of the results and conclusions of this
investigation, it is evident that there are a number of re-
lated conditions and refinements that are in need of pre-
sentation and application.
1. Additional research should be done utilizing ani-
mals higher on the phylo-genetic scale (e, g,, monkeys).
ko
This might allow for a more accurate assessment of intel-
lectual damage.
2, Future investigations should utilize a method of
accurately assessing and rigidly controlling the prevailing
biochemistry of each subject,
3, Future research should introduce a number of dif-
ferent levels of phenylalanine blood concentrations in an
attempt to establish a critical range during pregnancy.
4, PKU females desirous of pregnancy should return
to their low-phenylalanine diet before conception or soon
thereafter until further research demonstrates otherwise.
/
/
APPENDIX
MAZE DESIGN
Scalei 3/8"
"7T
I j I cj. |
t GOAL
in
BIBLIOGRAPHY
Books
Rugh, Roberts, Laboratory Manuel of Vertebrate Embryology. Minneapolis, Minn., Burgess Publishing Co., 1941-61,
Articles
Armstrong, M. D. and N. L. Low, "Phenylketonuria VIII, Re-lation Between Age, Serum Phenylalanine Level, and Phenylpyruvic Acid Excretion," Proceedings of the So-ciety for Experimental Biology and Medicine. XCIV (January, 1957) , 1^2-1^*57
Armstrong, M, E. and F. H, Tyler, "Studies on Phenylketonuria I. Restricted Phenylalanine Intake in in Phenylketonu-ria," Journal of Clinical Investment. XXXIV (March, 1955),'~5S5-580.
Berry, H. K., "Paper Chromotographic Method for Estimation of Phenylalanine," Proceedings of the Society for Ex-perimental Biology and Medicine. XCV (May, 195777 71-73»
Bickel, H., J. Gerrard, and E. M. Hickmans, "Influence of Phenylalanine Intake on Phenylketonuria," Lancet, II (October, 1953), 812-813.
Biel, W. C., "Early Age Differences in Maze Performance in the Albino Rat," Journal of Genetic Psychology, LVI (June, 19^0), 1^2-146.
Centerwall, W, R., R, F. Chinnock, and A. Pusavat, "Phenyl-ketonuria! Screening Programs and Testing Methods," American Journal of Public Health. L (November, i 9 6 0 ) , 1667-1677;
Centerwall, W. R. and S. A. Centerwall, "Phenylketonuria (Folling's Disease)! The Story of its Discovery," Journal of the History of Medicine and Allied Sciences. XVI (July, 1951), 192-295.
Centerwall, W, R. and C. A, Neff, HA Case Report of Children of Jewish Ancestry," Archives of Pediatrics. LXXVIII (October, 1 9 6 1 ) , 379-384.
^3
Centerwall, W. R., S. A. Centerwall, V. Armon, and L. B. Mann, "Phenylketonuria IIi Results of Treatment of Infants and Young Children. A Report of Ten Cases," Journal of Pediatrics, LIX (July, 1961), 102-118.
Clader, D. E., "Accelerated Intellectual Growth and Person-ality Development as Seen in Phenylketonuria Subjects During Medical Treatment," American Journal of Mental Deficiency. LXII (November, 1957)» 538-5^2.
Crome, L. and C, M. B, Pare, "Phenylketonuria! A Review and Report of the Pathological Findings in 4 Cases," J ournal of Mental Science, CVI (April, i 9 6 0 ) , 862-863.
Davison, A, N. and M. Sandler, "Inhibition of 5-Hydroxytryp-tophan Decarboxylase by Phenylalanine Metabolites," Nature (London), CLXXXI (January, 1958)* 186-187.
Davison, A. N. and J, Dobbing, "Myelination as a Vulnerable Period in Brain Development," British Medical Bulletin. XXII (January, 1966) , ^0-^4. .
Foiling, A., "Phenypyruvic Acid as a Metabolic Anomaly in Connection with Imbecility*" NORD. MED. TIDSKR.. XIII (March, 193*0. 105^-1059.
Guthrie, R. and A. Susi, "A Simple Phenylalanine Method for Detecting Phenylketonuria in Large Populations of Newborn Infants," Pediatrics. XXXII (September, 1963) , 338-3^3.
Hanson, A., "Inhibition of Brain Glutamic Acid Decarboxylase by Phenylalanine Metabolites," Naturwissenschafte. VL (October, 1958)» ^23.
Henry, R. J.t C. Sobel, and N. Chiamori, "Method for Deter-mination of Serum Phenylalanine with Use of the Kapeller-Adler Reaction," American Medical Association Journal of Diseases of Children.VCVI (December. lQ^7^f
Horner, F. A. and C. W. Streamer, "Effect of a Phenylalanine-Restricted Diet on Patients with Phenylketonuria; Clini-cal Observations in Three Cases," Journal of the Ameri-can Medical Association. CLXI (August, 195£T,"T528-l63b.
Jervis, G. A., "Phenylpyruvic Oligophreniaj Introductory Study of 50 Cases of Mental Deficiency Associated with Excretion of Phenylpyruvic Acid," Archives of Neuroloev Hid Psychiatry. XXXVIII (November,' 1937), 9^.963.
44
Kang, E, and R. S, Paine, "Elevation of Plasma Phenylalanine Levels During Pregnancies of Women Heterozygous for Phenylketonuria," Journal of Pediatrics. LXIII (August, 1963), 283-289.
Kerr, G. R. and H. A, Waisman, "Phenylalanine! Transplacen-tal Concentrations in Rhesus Monkeys," Science, CLI (May, 1965). 685-695.
Knox, W, E,, "An Evaluation of the Treatment of Phenylketonu-ria with Diets Low in Phenylalanine," Pediatrics. XXVI (July, i960), 1-11.
Koch, R., K, Fishier, S. Schild, and N. Ragsdale, "Clinical Aspects of Phenylketonuria," Mental Retardation. II (February, 196^), 47-5^.
LaDu, B, N,, and P. J, Michael, "An Enzymatic Spectrophoto-metry Method for the Determination of Phenylalanine in Blood," J ournal of Laboratory and Clinical Medicine. LV (March, i960), ^91 !
Langdell, John I., "Phenylketonuriai Childhood and Adoles-cence," Current Psychiatric Therapies. V (1965), ^9-56.
Mabry, C. C., J. C. Denniston, T. L. Nelson, and C, D. Son, "Maternal Phenylketonuria! A Cause of Mental Retarda-tion in Children Without the Metabolic Defect," New England Journal of Medicine. CCLXIX (December. 10?/^ . 1404-1 *1-08.
Mabry, C. C., J, C. Denniston, and J. G. Caldwell, "Mental Retardation in Children of Phenylketonuria Mothers," New England Journal of Medicine. CCLXXV (December. 1936), 1331 -13 3FI
McCaman, M, W. and E. Robins, "Fluorimetric Method for the Determination of Phenylalanine in Serum," Journal of Laboratory and Clinical Medicine. LIX (Mav. 1 Q ^ . 885-890.
Neame, K, D., "Phenylalanine as Inhibitor of Transport of Amino Acid in^Brain," Nature (London), CIIIV (October,
Paine, R. S., "The Variability in Manifestations of Untreated Patients With Phenylketonuria (Phenylpyruvic Aciduria)," Pediatrics. XX (August, 1957). 290-302.
Pare, C. M. B,f M. Sandler, and R. S. Stacey, "Decreased 5-Hydroxytryptophan Decarboxylase Activity in Phenyl-ketonuria," Lancet, II (November, 1958)» 1099-1101.
Penrose, L, S., "Inheritance of Phenylpyruvic Amentia (Phenyl-ketonuria)," Lancet. II (July, 1935)» 192-19^.
Penrose, L. A, and J. H. Quastel, "Metabolic Studifes on Phenylketonuria," Biochemical Journal, XXXI (February, 1937), 266-2?^.
Polidora, V. J,, D, E, Boggs, and H, A, Waisman, "A Behavior-al Deficit Associated With Phenylketonuria in Rats," Proceedings of the Society for Experimental Biology and Medicine, CXIIl (September, 1963)» 817-820.
Schalock, R. L. and F. D. Klopfer, "Phenylketonuria! Endur-ing Behavioral Deficits in Phenylketonuria Rats," Science. CLV (February, 1967)» 1033-1035.
Stevenson, R. E. and C. C. Huntley, "Congenital Malforma-tions in Offspring of Phenylketonuria Mothers," Pedi-atrics, XL (July, 1967), 33-^5.
Thompson, W. R, and K, Kano, "Effects on Rat Offspring of Maternal Phenylalanine Diet During Pregnancy," Journal of Psychiatric Research. Ill (May, I965K 91-98.
Waisman, H.' A. and H. F. Harlow, "Experimental Phenylketonuria in Infant Monkeys," Science. CIIIL (February, 1965), 685-695.
Woolf, L. I., R, Griffiths, and A. Moncrieff, "Treatment of Phenylketonuria With a Diet Low in Phenylalanine," British Medical Journal. I (January, 1955), 56-6*1-,
Reports
Bickel, H., Chapter in Phenylketonuria. edited by Frank. Lyman, and Charles C. Thomas, Springfield, 111., 1963.
R Cymogram . Allen and Hanbury's Ltd., Bethnal Green, London,
E. I., England.
Guthrie, R. and S. Whitney, "Phenylketonuria! Detection in the Newborn Infant as a Routine Hospital Procedure," Children's Bureau Publication. No. Ul9, U. S. Depart-ment of Health, Education, and Welfare, Washington, D. C., 20201, 1964.
R Ketonil . Merck, Sharp and Dohme, Philadelphia,,Pa,
R LoffiH&l&C - MadH ,T AKM n AM ft*. 1 1 -1
k6
MinafenR, Trufood Ltd., 113 Newington Causeway, London, S. E. I., England•
R Phenistix , Ames Company, Inc. Elkhart, Ind,
