HYPERVALINEMIA
A
Defect
in Valine
Transamination
Joseph Dancis, Joel Hutzler, Keiya Tada, Yoshiro Wada,
Toshio Morikawa, and Tsuneo Arakawa
From the Departments of Pediatrics, New York University School of Medicine,
and the University of Tohokti School of Medicine
(Received September 23; accepted for publication November 29, 1966.)
J.D. is a career investigator, The National Institute of Child Health and Human Development,
K6-GM-16,710-03. ADDRESS: New York University School of Medicine, 550 First Avenue, New York, New
York 10016.
Aided by grants from The National Foundation and the National Institute of Child Health and
Human Development,
#
PHS-HD-00462-1 1A1.PEDIATRICS, Vol. 39, No. 6, June 1967
ARTICLES
811
A
PREVIOUS publicationi described a2-month-old Japanese infant who
started to vomit shortly after birth, failed to
gain weight, and appeared to be mentally
retarded. Biochemical studies revealed an elevation of valine in the urine. The admin-istration of valine by mouth produced a prolonged elevation of plasma valine as compared to normal subjects, without a concomitant increase in a-keto acids.’ This suggested a reduction in the capacity to de-grade valine, probably at the initial step in the metabolic pathway.
In the present study, the metabolism of
valine has been investigated in this same patient, now 3 years old, using the
periph-eral leucocyte. The patient had been pre-viously studied with the technique original-ly developed for the investigation of maple syrup urine disease3’ with inconclusive results. The technique has now been
modified to detect the formation of keto
acids more precisely, as an indicator of transamination. The white cells were incu-bated with 14C-labeled amino acids as pre-viously described.3” To the incubation medi-um was added the specific keto acid to
“trap” any radioactive keto acids produced
by the leucocytes. Using this method, a de-fect in the transamination of valine has been confirmed.
The metabolism of valine by placenta from a normal term pregnancy was also in-vestigated to determine if the metabolic de-fect might be studied in this tissue. Active
formation of the keto acid was
demon-strated.
METHODS
The white blood cells were harvested
following sedimentation with fibrmnogen.
Plasma containing white blood cells from
approximately 2 ml of blood was placed in
each of the incubation flasks and
centri-fuged. The supernatant was decanted and
replaced with the following medium: 0.95 ml Krebs-Ringer phosphate buffer, pH 7.4, 0.25 Mole DL-valine-1-14C (1.0 p.c), 0.5 .tMole sodium -keto isovalerate, total vol-ume 1.0 ml.
Incubation was for 75 minutes at 37#{176}C
with agitation. Leucocytes were also
incu-bated with equivalent amounts of DL-leu-cine-1-14C and sodium z-keto isocaproate. Control flasks containing medium includ-ing the radioactive amino acid, but
with-out leucocytes, served as reagent blanks.
Each flask had a center well containing 0.1 ml of 1% sodium carbonate in 0.5N po-tassium hydroxide to trap the radioactive carbon dioxide liberated by decarboxyla-tion. All tests were run in duplicate.
At the end of the incubation, one drop of iN hydrochloric acid was added to the medium. The leucocytes were removed from the incubation medium by
centrifu-gation, and to the supernatant was added
‘4C, DL-phenylalanine-i-14C, (4 ,.Moles,
0.4 p.c), L-isoleucine-U-1’C, or
L-methio-nine-methyl-’ ‘C (2 .Moles, 0.2 p.c). To each
flask were added 1 .Mole of the respective keto acid, and pyridoxammne .2HC1 (10 p.g)
in Krebs-Ringer phosphate buffer. The
re-maining procedure was as described here.
The contents of the center well were
transferred quantitatively to 18 ml of scin-tillation counting solution for the deter-mination of radioactivity. The solution con-tained 500 ml toluene, 250 ml methanol, 250 ml phenylethylamine, and the
scintil-lants, 5.5 gm PPO (2,5-diphenyloxazole)
0.11 gm dimethyl POPOP
(1,4-bis-2-[4-methyl-5-phenyloxazolyl] -benzene).
Radioactivity was determined in a Pack-ard TriCarb liquid scintillation counter.
MATERIALS
The radioactive amino acids were ob-tained from New England Nuclear
Corpo-ration, Boston, Massachusetts. Sodium
-TABLE I
TIIANSAMINATION AND 1)Ec’ARBoxYLATloN BY
PERIPHEIIA L LEU(OCYTES
Subject Leucine Keto (‘02 Acid J‘aline , Acid Ilypervalinemia Controls 1 3 4 36,8O 30,040 3,300 1,450 49,30 1,715 50 749 193 1,760 0 7,65 7,I’20 6,40 9,180 153 338 340 H7 786
Leucocytes were incubated with 0.5 ,uMole (1.0 /Ac) of DL-leucine-1-’4C or DL-valine-1-’4C and the
radio-active keto acids and CO2 were determined. Results
are the average of duplicate determinations in
dis-integrations per minute.
Valine Metabolism in Placenta
A term placenta was obtained, refrig-c-rated immediately after delivery, and tested within 2 hours. Fragments of tissue
were taken from the central substance of
the placenta to avoid sampling of the ma-ternal decidual plate. Four flasks were set up-two with 20 mg each and two with 100 mg each of placental tissue. The in-cubation medium was 0.6 ml Krebs-Ringer
phosphate buffer, pH 7.4 containing 2.2 Mole DLvaline114C (0.2 p.c) and 1.0 p.Mole sodium 2-keto isovalerate acid.
In-cubation was for 1 hour at 37#{176}C,under
air. Analysis for the formation of
radio-active keto acids was as described here.
Selection of Patients
The control subjects were Japanese,
drawn from the same community as the patient.
RESU LTS
The first series of experiments
demon-strated that the metabolism of leucine by
the patient and the control subjects is quite similar (Table I). In contrast, incuba-tion of the patient’s leucocytes with valine-1-14C failed to produce any detectable
ra-dioactive keto acids. However, there is
some ‘4C02 produced.
The second series of experiments (Table II) provides evidence that, despite the de-fect in valine transamination, the patient’s leucocytes can transaminate other amino acids.
BLOCK IN HYPERVALINEMIA
NH
I
NHCH3 12 3
C C COOH
CH3H H
VALINE
0
CH3-. ii
- C
-
C-
000HCH3- H
H H
CH3-C -C-C--COOH H CH3H
ISOLEUCINE
NH
CH3 H 12
C-C-C-000H
CH3-’H H H
NH3
NH
CH3 H
C-C
-
C -COOHCH3H H
R-COOH
±
Co2
BLOCK IN
MAPLE SYRUP
URINE DISEASE
LEUCINE
Fic. 1. Degradative pathway of leucine, isoleucine, and valine. The metabolic block in hypervalinemia
involves the transamination of valine alone. In maple syrup urine disease, the oxidative decarboxylation
of all three branched-chain keto acids is defective.
ARTICLES
H H
CH3- C-C-C-COOH
H OH3
contained an average of 954 disintegra-tions per minute in the keto acid fraction; the flasks with 100 mg of tissue contained 3,849 disintegrations per minute.
COMMENT
The first step in the degradation of
amino acids is generally the removal of the
amino group with the formation of the
corresponding keto acid (Fig. 1). This may
be accomplished by oxidative-deamination,
or by the transfer of the amino group to an acceptor, usually a-ketoglutarate. The latter mechanism appears to be the pre-dominant one in mammalian tissue. In the course of investigating maple syrup urine
disease, the transfer of the amino group
from the branched chain amino acids (ic-u-ci, isoleucine, and valine) to a-ketoglu-tarate has been demonstrated with ho-mogenates of human tissue obtained at autopsy.9
The enzyme defect in this patient is evi-dently the transaminase for valine. Cir-cumstantial evidence had been provided by the demonstration of hypervaliriemia in
the absence of an excess excretion of the keto acid.1 The investigation of the periph-eral leucocyte provides direct evidence of the deficiency in transamination (Table I). The radioactive CO2 liberated from the valine, in the absence of radioactive keto acids, probably represents degradation through an alternate, minor pathway.
TABLE II
TRANSAMINATION OF SELECT AMINO ACIDS BY
PATIENT AND CONTROL SUBJECTS
Isoleucine 346 268 222
Leucine 387 183 143
Methionine 198 98 55
Phenylalanine 48 32 42
Leucocytes were incubated with one of the following
substrates: 4 iMoles (0.4 j.ic) of DL-valine-1-’4C, 1)L-Ieucine-1-#{176}C, DL-phienylalanine-l-#{176}C, or 2 MoIes (0.2 j.c) of L-isoleucine-U-’4C or L-methiionine-Inethyl #{176}C.The radioactive keto acids are reported in
more than one amino acid, though there is some opinion to the contrary.8’1#{176} The pa-tient with hypervalinemia provides strong
evidence that there is a specific
trans-aminase for valine in the human.
The elevation of only valine in the se-rum indicated that the degradation of other amino acids was normal despite the
deficiency of valine transamination, and
that the presence of other transaminases
was not adequate for the catabolism of valine. More direct evidence for the spe-cificity of the valine transaminase was
sought in the peripheral leucocyte using
four amino acids in addition to valine. Leucine and isoleucine were chosen be-cause of their structural similarity to va-line. The transamination of phenylalanine represents, under normal circumstances, a minor pathway so that a deficiency might not have been reflected in an elevated
plasma level. The relative importance of
transamination in the catabolism of methi-onine is not completely clear.
The results in Table II further support the conclusion that the defect in hyper-valinemia is in an enzyme specific for va-line transamination. The observed radio-activity in Table I is considerably higher than in Table II. This may be largely ac-counted for by the differences in substrate concentrations, higher substrate concentra-tions being introduced in the second series of experiments to better quantitate the en-zyme capacities. Tile radioactivity in the keto acids formed from phenylalanine is low, but the results are consistent and
ap-Preciably above the reagent blanks which
averaged less than five disintegrations per mmute above background.
In contrast to hypervalinemia where one amino acid is elevated in the plasma, all
common enzyme for all three branched-chain keto acids is “illogical” in terms of evolutionary concepts and survival value, and that there are probably individual de-carboxylases. However, a specific enzyme at the first degradative step could provide an opportunity for the control of the rate of degradation of each amino acid, making such specificity of control less necessary in the second step.
It is not possible to state whether tile metabolic defect in hypervalinemia is ge-netic in origin. This patient represents the only case of hypervalinemia reported so far. There are no siblings. The parents ap-pear normal and are not consanguineous.
Load tests have failed to demonstrate a
partial defect.2
In a previous study2 the child was placed on a low valine diet for a short period of observation. The serum valine level re-turned to normal, vomiting ceased, hyper-kinesia decreased, and there was a rapid gain in weight. However, it is not certain that the mental retardation is related to the metabolic error. The practice of searching for amino acid dyscrasias in patients who are mentally defective greatly increases tile likelihood of coincidence.
It was of additional interest to determine if the metabolic anomaly could be demon-strable in the placenta. Earlier studies have demonstrated the presence of an alanine-glutamic acid transaminase in human placenta.1’ With the use of the techniques
described here, it has been possible to de-tect with ease the formation of
-ketoiso-valeric acid from valine11C with 20 mg of
placental tissue. The placenta is almost
en-tirely fetal in origin and the synthesis of
the same genetic control as that in the ieucocyte. If this proves true, it may be pos-sible to make the diagnosis before birth by needle biopsy of the placenta, a technique
that has been used successfully by Alvarez
for pathological studies, even in early preg-nancies.’3
SUMMARY
The metabolism of the peripheral leuco-cyte has been studied in a child presenting with mental retardation and hypervalin-emia. A defect in the transamination of valine was demonstrated. Evidence is pre-sented that the transamination of value is dependent on an enzyme that is specific for valine. Transamination of valine is demon-strable in placenta, suggesting the possibil-ity that the diagnosis may be made before birth.
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