Free Radical Biology & Medicine, Vol. 32, No. 9, pp.

906 –911, 2002

Copyright © 2002 Elsevier Science Inc.
Printed in the USA. All rights reserved
0891-5849/02/$–see front matter

PII S0891-5849(02)00781-5

Original Contribution
OXIDATIVE STRESS IN A PHENYLKETONURIA ANIMAL MODEL

NURAN ERCAL,* NUKHET AYKIN-BURNS,* HANDE GURER-ORHAN,* and J. DAVID MCDONALD†

*University of Missouri-Rolla, Department of Chemistry, Rolla, MO, USA; and †Wichita State University, Department of
Biological Sciences, Wichita, KS, USA

(Received 3 December 2001; Accepted 8 February 2002)

Abstract—Oxidative stress is seen in various metabolic disorders for unknown reasons. Oxidative stress is defined as
an imbalance between pro-oxidant and antioxidant status in favor of the former. This study investigated whether
oxidative stress exists in phenylketonuria (PKU) using the BTBR-Pahenu2 animal model for PKU. Animals (14 –24
weeks old) were sacrificed and brain and red blood cells (RBCs) were obtained aseptically. The lipid peroxidation
by-product, evaluated as malondialdehyde (MDA), was significantly higher in the brains and RBCs of PKU animals
(n ⫽ 6) than in controls (n ⫽ 6). Glutathione/glutathione disulfide, a good indicator for tissue thiol status, was
significantly decreased both in the brains and RBCs. Some antioxidant enzymes were also analyzed in RBCs, including
glucose-6-phosphate dehydrogenase (G6PD), which provides the RBC’s main reducing power, reduced nicotinamide
adenine dinucleotide phosphate (NADPH), and catalase detoxifies H2O2 by catalyzing its reduction to O2 and H2O. Both
catalase and G6PD were significantly increased in the RBCs of PKU animals. © 2002 Elsevier Science Inc.

Keywords—Phenylketonuria, Oxidative stress, Glutathione, Free radicals

INTRODUCTION it yet. Recent studies indicate that oxidative stress might

play a role in PKU pathogenesis.
Phenylketonuria (PKU) is an inborn error of amino acid Oxidative stress is commonly observed in some in-
metabolism caused by a deficiency of phenylalanine hy- born errors of intermediary metabolism [4 –7]. Although
droxylase (PAH; E.C. 1.1416.1) [1]. PAH hydroxylates the reason for this oxidative stress is not completely
phenylalanine (Phe), converting it to tyrosine (Tyr). Un- understood, it may be caused by the accumulation of
der conditions of PAH deficiency, Phe cannot be con- toxic metabolites that lead to the excessive production of
verted to Tyr. Phe accumulates and the concentrations in free radicals. It may also be that an unusual increase in
plasma may reach millimolar levels [2]. At these very metabolic by-products will directly, or indirectly, deplete
high levels, some of the accumulated Phe can then be a cell’s antioxidant capacity.
metabolized by alternative pathways making phenylk- When a cell’s pro-oxidants exceed its antioxidant
etones such as phenylpyruvate, phenyllactate, and phe- capacity, free radicals accumulate and oxidative stress
nylacetate. Consequently, this metabolic disorder was occurs. Various inborn errors of amino acid metabolism
given the name, “phenylketonuria,” to emphasize this show an increased formation of free radicals. For exam-
increase in phenylketones. If left untreated, PKU causes ple, in tyrosinemia Type 1, liver glutathione (GSH) lev-
severe mental retardation. Its treatment is rather difficult els were found to be significantly decreased [8]. Ty-
and involves a very strict diet, probably for life [3]. rosinemia Type 1 is caused by a deficiency of
Many studies have elucidated the mechanisms underly- fumarylacetoacetatase, the enzyme that catalyzes the last
ing mental retardation from PKU but, unfortunately, no step of the catabolic pathway of Tyr. As a result of this
single factor has been identified as being responsible for deficiency, the highly reactive compounds maleylacetate
and fumaylacetoacetate are formed. When these com-
pounds were administered to animals, GSH levels in
Address correspondence to: Nuran Ercal, Department of Chemistry, their livers were significantly depleted [9]. GSH plays a
University of Missouri-Rolla, 142 Schrenk Hall, Rolla, MO 65409-
0010, USA; Tel: (573) 341-6950; Fax: (573) 341-6033; E-Mail: major role in the first-line antioxidant defense of cells
nercal@umr.edu. against pro-oxidants and, therefore, depletion of GSH in

906
 Phenylketonuria and oxidative stress 907

tyrosinemia Type 1 suggests a free radical involvement plasma Phe level and normal behavior under normal
in tyrosinemia Type 1. holding conditions. The homozygous mutant BTBR-
In treated PKU patients, studies [10,11] also show that Pahenu2 mouse is a counterpart of the human PKU. These
there is oxidative stress due to a deficiency in selenium animals show no residual activity of the liver PAH along
(Se), one of the important antioxidants in cells [10]. with very high plasma Phe levels [13]. Twelve animals
However, another study reported a significant decrease in (6 BTBR-Pahenu1 as control, 6 BTBR-Pahenu2 as PKU
red blood cells (RBCs) glutathione peroxidase (GPx) animal model), ranging in age from 14 to 24 weeks old,
activity in PKU and mild hyperphenylalaninemia (m- were sacrificed under anesthesia. Blood samples were
HPA) patients, even though their Se levels were normal- collected via intracardiac puncture. Plasma and the buffy
ized with Se supplementation [4]. Furthermore, Phe- coat were removed by centrifugation for 10 min at
restricted diets are enriched with vitamins, minerals, and 1000 ⫻ g. The RBCs were washed three times with an
trace elements to achieve their recommended dietary equal volume of cold saline. The RBC samples were
intake levels. However, in PKU infants and children low processed immediately for measurement of G6PD, CAT,
plasma retinol and zinc concentrations have been re- GSH/GSSG. An aliquot of RBCs were kept at ⫺70°C
ported along with iron depletion, appeared as low serum and analyzed in 48 h for MDA levels. Tissue samples
ferritin concentrations indicating that inborn errors of were collected after the animals were sacrificed and kept
amino acid metabolism may reduce the bioavailability of at ⫺70°C until analysis.
some essential micronutrients and antioxidants and in-
duce oxidative stress [12]. The present study was under-
taken to evaluate the oxidative status of PKU animals Determination of phenylalanine
that were fed a regular diet ad libitum. The diet contains Plasma Phe levels were measured spectrofluorometri-
all the necessary nutrients including minerals (Ca, P, K, cally based on the method of McCaman and Robins [14]
Na, Mg, Fe, Mn, Zn, Se, Cu) and vitamins (␣-tocopheryl by using Phe determination kit from Sigma Chemical Co.
acetate, vitamin A palmitate, vitamin B12, thiamin, ribo- Phe reacts with ninhydrin in the presence of copper ions
flavin, niacin, folic acid, vitamin D3, vitamin K). The to form a highly fluorescent complex, which is propor-
status of oxidants in their brains and RBCs was evaluated tional to Phe concentration.
by malondialdehyde (MDA; for lipid peroxidation),
GSH/glutathione disulfide (GSSG) (main thiol status)
and some antioxidant enzymes (glucose-6-phosphate de- Determination of GSH and GSSG
hydrogenase [G6PD] and catalase). Because results
GSH and GSSG concentrations were determined by
showed that there was oxidative stress, mechanisms that
using the method developed in our laboratory [15].
may cause this ongoing oxidative stress are discussed.
Briefly, free sulfhydryl groups were reacted with N-(1-
pyrenyl)-maleimide and subsequently separated and de-
MATERIALS AND METHODS
tected by reverse phase high-performance liquid chroma-
tography (HPLC) with fluorescence detection. GSSG
Chemicals
was derivatized as GSH after initial reduction by gluta-
The N-(1-pyrenyl)-maleimide, 1,1,3,3-tetrame- thione reductase and reduced nicotinamide adenine dinu-
thoxypropane, and 2-vinyl pyridine were purchased from cleotide phosphate (NADPH) following free thiol alky-
Aldrich (Milwaukee, WI, USA). All other chemicals lation by 2-vinylpyridine.
were obtained from Sigma (St. Louis, MO, USA).
MDA analysis by HPLC
Animals
The samples were analyzed according to the Draper
The PAH mutation in BTBR-Pah ENU1 and ENU2 method [16]. In brief, MDA was derivatized by thiobar-
strains was produced (over 20 generations ago) by ENU bituric acid (TBA). Following derivatization, the sample
mutagenesis technique detailed in Sarkissian et al. [13]. was extracted with butanol and analyzed by reverse
Wild type mice (BTBR/Pas background) were treated phase HPLC with fluorescence detection.
with an alkylating agent (N-ethyl-N⬘-nitrosurea: ENU) to
induce mutations on the Phe hydroxylase locus (Pah). Determination of G6PD activity
The homozygous mutant BTBR-Pahenu1 mouse is a
counterpart of the human non-PKU-hyperphenylalanyl- Determination of G6PD activity was performed using
emia (non-PKU-HPA). Although it has a missense mu- a spectrophotometer as detailed in Tietz, where glucose-
tation in the Pah gene, this mouse has both a normal 6-phosphate and NADP⫹ were used as substrates [17].
908 N. ERCAL et al.

Table 1. Oxidative Stress Parameters of Red Blood Cells from PKU Table 2. Oxidative Stress Parameters of Brain Tissue from PKU
Animal Model Animal Model

GSH GSSG MDA GSH GSSG MDA

(nmol/g Hb) (nmol/g Hb) GSH/GSSG (nmol/g Hb) (nmol/g (nmol/g (nmol/100 mg
protein) protein) GSH/GSSG protein)
Control 23.91 ⫾ 1.02 3.43 ⫾ 0.26 7.07 ⫾ 0.67 37.36 ⫾ 3.04
PKU 21.24 ⫾ 1.24† 5.91 ⫾ 1.30† 3.73 ⫾ 0.82‡ 43.55 ⫾ 7.48* Control 18.68 ⫾ 1.63 3.40 ⫾ 0.57 7.07 ⫾ 0.67 18.54 ⫾ 3.26
PKU 15.28 ⫾ 1.15* 5.69 ⫾ 0.72† 5.18 ⫾ 0.62‡ 35.89 ⫾ 3.27‡
* p ⬍ .05 compared to corresponding value for control group.
†
p ⬍ .005 compared to corresponding value for control gorup. * p ⬍ .005 compared to corresponding value for control group.
‡
p ⬍ .0001 compared to corresponding value for control group. †
p ⬍ .0005 compared to corresponding value for control gorup.
‡
p ⬍ .0001 compared to corresponding value for control group.

Catalase assay
GSH, GSSG, and MDA concentrations in brains
Catalase activity was determined spectrophotometri- Effects of PKU on brain oxidative stress parameters
cally, based on the disappearance of 10 mM H2O2 at 240 are summarized in Table 2. PAH deficiency caused a
nm in the presence of catalase [18]. significant decrease in GSH (p ⬍ .005) and a significant
increase in GSSG (p ⬍ .0005) levels in the brain samples
compared to the control group. Lipid peroxidation was
Hemoglobin and protein determination highly induced in the brains of PKU animals. MDA
levels in brain tissues of the PKU and control groups
Hemoglobin contents of the RBC samples were mea- were found to be statistically different (p ⬍ .05).
sured spectrophotometrically, as detailed by Tietz [17].
Protein levels were measured using Coomassie Blue
(Bio-Rad, Hercules, CA, USA) according to Bradford G6PD and catalase activity in RBCs
[19].
Catalase (Fig. 1) and G6PD (Fig. 2) activities were
also measured in RBCs. For both, it was found that
Statistical analysis catalase and G6PD had significantly increased (p ⬍
.0005) in RBCs of PKU animals, as compared to their
The one-way analysis of variance test was used to control. Brain catalase activity was undetectable.
analyze the significance of the differences between the
control and experimental groups. DISCUSSION

RESULTS PKU is a biochemical genetic disorder caused by a

mutant allele at the Pah locus that results in PAH defi-
ciency. Because PAH is required to convert Phe into Tyr,
Plasma phenylalanine levels
a PAH deficiency causes an accumulation of Phe and its
Phe levels in the plasma of control and PKU animals alternative metabolic pathway products. If untreated, pa-
were found as 90.9 ⫾ 30.3 and 1399.9 ⫾ 418.1 ␮M,
respectively. These values are comparable with those
that are seen in human PKU patients.

GSH, GSSG, and MDA concentrations in RBCs

Table 1 displays the selected oxidative stress param-

eters in the RBCs of the control and experimental groups.
GSH, GSSG levels and, consequently, GSH/GSSG ratios
in RBCs showed significant differences (p ⬍ .005) in
PKU animals compared to the corresponding control
group. MDA was used as the main criteria for the lipid
peroxidation and it was found that lipid peroxidation in
Fig. 1. Catalase activity in RBCs. Catalase activity was determined
RBCs was increased in PKU animals compared to con- spectrophotometrically, based on the disappearance of 10 mM H2O2 at
trol groups (p ⬍ .05). 240 nm in the presence of catalase (15).
 Phenylketonuria and oxidative stress 909

levels could inhibit the cell membrane’s other amino acid

transportation systems [24]. As a result, general protein
synthesis has been reported to decrease in cells [25].
When GSH is decreased, cells enter a low-antioxidant
mode and are more prone to oxidative damage caused by
free radicals. This is especially true for cells which are
innately more vulnerable to oxidative attacks, such as
RBCs and brain cells. Membrane lipids can then be
easily oxidized in cells in a low-antioxidant mode. MDA
is a by-product of lipid peroxidation reaction [26]. MDA
analysis, which is widely used to detect lipid peroxida-
Fig. 2. G6PD activity in RBCs. G6PD activity was determined using a tion in tissues, has received much criticism because of its
spectrophotometer as detailed in Tietz [17], where glucose-6-phosphate
and NADP⫹ were used as substrates (14). nonspecific aspects [27]. Nevertheless, it is one of the
most commonly used techniques. In our study, MDA-
TBA adduct was identified by HPLC. HPLC identifica-
tients with classical PKU without dietary restrictions tion of MDA-TBA adduct has an advantage over the
may become mentally retarded. Unfortunately, the bio- thiobarbituric acid reactive substances (TBARS) method,
chemical mechanisms of brain dysfunction are not which is not specific for lipid peroxidation. As shown in
known with certainty, although recent studies have Tables 1 and 2, MDA levels were significantly higher in
shown that oxidative stress seems to be involved in PKU RBCs and brain tissue of PKU animals. Wilke et al.
[4 –7]. It is well-known that brain tissue is very vulner- measured the plasma MDA levels in the control and
able to oxidative damage because it does not have a PKU children and found that levels were significantly
strong antioxidant defense system. It has been previously higher in PKU children than in the control group [28].
suggested that phenyl-ketone metabolites are toxic to a The G6PD enzyme, another antioxidant parameter in
developing brain [20]. Oxidative stress may also aug- RBCs, is involved in the first committed step in the
ment their toxicity, although there is no evidence indi- pentose phosphate pathway and supplies NADPH, an
cating a relationship between mental retardation and important source of reducing power of RBCs. G6PD is
oxidative stress. regulated by NADPH/NADP⫹ ratio [29]. When cells are
Oxidative stress is defined as an increase in pro- exposed to free radicals, this ratio decreases, demanding
oxidative damage to cells due to an increase in free more NADPH. Therefore, an increase in G6PD enzyme
radical production or impairment in antioxidant defense. indicates that cells are under oxidative stress.
Several parameters are used to assess oxidative status of Like the G6PD, the activity of catalase, another anti-
a cell, with levels of GSH and GSSG commonly being oxidant enzyme, was determined in RBCs and was found
used as oxidative stress parameters [21]. GSH, ␥-glu- to be significantly increased. The catalase enzyme is
tamyl-cysteinyl-glycine, is regarded as the main antiox- involved in the formation of O2 and H2O from H2O2
idant arsenal of a cell, therefore GSH is continuously [30]. Although H2O2 is not considered to be a free
synthesized by the ␥-glutamyl cycle [22]. GSH becomes radical, it can easily form superoxide and hydroxyl rad-
a very important antioxidant for cells that are more icals. Removal of H2O2 by catalase is one of the most
vulnerable to oxidative damage, such RBCs, although important antioxidant systems, and alterations in catalase
RBCs innately contain higher levels of GSH [23]. The activity in tissues indirectly indicate a possible oxidative
present study shows that these levels decrease in RBCs stress situation.
and brain tissues of PKU animals, whereas GSSG levels The data from the present study indicate that there is
increase compared to the control group, indicating oxi- a disruption of pro-oxidant/antioxidant balance in PKU
dative stress. Sierra et al. measured the total glutathione animals developed genetically by McDonald et al. [31].
(GSH⫹GSSG) in RBCs obtained from PKU and m-HPA To the best of our knowledge, this is the first study to
patients [4]. Although they did not report the data, they investigate whether there is oxidative stress in PKU
found no difference in total GSH levels. In another study, animals. All previous studies of oxidative stress involve-
PKU patients with lower erythrocyte GSH values as ment in PKU were performed by studying PKU children
compared to the control group have been reported [7]. or adults. Because they were on a strict diet, a decrease
Both studies were performed on human patients treated in their antioxidant intake, especially Se, was found to be
with Phe restricted diet. A decrease in GSH could be due responsible for the oxidative stress seen in PKU. A
to either an inhibition created by high Phe on the amino recent study showed no decrease in plasma levels of Se,
acid transport system for Cys and Gly, or by an increase even though the GPx activity in RBCs of PKU patients
in free radical formation. It was shown that high Phe was significantly decreased [4].
910 N. ERCAL et al.

This observed oxidative stress in PKU could be due to that antioxidant supplementation should be revised in
many causes: (i) As others have suggested, decreased PKU diets to prevent possible further damage from free
antioxidant intake or deficient antioxidant metabolism radicals.
could result in oxidative stress in PKU. Se was shown to
be decreased and, because of Se deficiency, GPx activity Acknowledgements — The authors would like to thank Barbara Harris
was found to have decreased also, because Se is a co- and Stephanie Maiden for their secretarial assistance in the preparation
factor for GPx enzyme. Because GPx removes toxic of this manuscript and Patricia Kulcharyk for her technical help.
peroxides, toxic peroxides tend to accumulate when there
is insufficient GPx. (ii) Phe alternative pathway products
REFERENCES
may induce formation of free radicals. Phe is normally
[1] Scriver, C. R.; Kaufman, S.; Eisensmith, R. C.; Woo, S. L. C. The
found in micromolar concentrations in the plasma and phenylalaninemias. In: Scriver, C. R.; Beaudet, A. L.; Valle, D.;
tissues. If, however, PAH is missing, as in PKU, Phe Sly, W. S., eds. The metabolic and molecular bases of inherited
accumulates and can reach millimolar concentrations in disease. New York: McGraw-Hill; 1995:1015–1075.
[2] Krause, W.; Halminski, M.; McDonald, L.; Demure, P.; Salvo, R.;
PKU patients. When the normal metabolic pathway of Friedes, S. R.; Elsas, L. Biochemical and neuropsychological
Phe is blocked, it goes through alternative pathways that effects of elevated plasma phenylalanine in patients with treated
are normally inactive. Metabolic end products of these phenylketonuria. J. Clin. Invest. 75:40 – 48; 1985.
[3] Smith, I.; Wolff, O. H. Natural history of phenylketonuria and
pathways could induce free radical formation. It was influence of early treatment. Lancet 2:540 –544; 1974.
shown that most of the alternative Phe pathway products [4] Sierra, C.; Vilaseca, M. A.; Moyano, D.; Brandi, N.; Campistol,
are toxic [32], although their oxidative toxicity was not J.; Lambruschini, N.; Cambra, F. J.; Deulofeu, R.; Mira, A.
Antioxidant status in hyperphenylalaninemia. Clin. Chim. Acta
specifically investigated. (iii) Phe could also serve as a 276:1–9; 1998.
substrate for Tyr hydroxylase enzyme [1]. Because Phe [5] Artuch, R.; Vilaseca, M. A.; Moreno, J.; Lambruschini, N.; Cam-
is extremely high and Tyr is very low, the less discrim- bra, F. J.; Campistol, J. Decreased serum ubiquinone-10 concen-
trations in phenylketonuria1,2. Am. J. Clin. Nutr. 70:892– 895;
inative Tyr hydroxylase acts on Phe, making dihydroxy- 1999.
phenylalanines. Oxidation of these catecholamines can [6] Fisberg, R. M.; Silva-Femandes, M. E.; Fisberg, M.; Schmidt,
produce superoxide and hydrogen peroxide in a very B. J. Plasma zinc, copper, and erythrocyte superoxide dismutase
in children with phenylketonuria. Nutrition 15:449 – 452; 1999.
complicated series of reactions [33]. (iv) Another reason [7] van Bakel, M. M. E.; Printzen, G.; Wermuth, B.; Wiesmann,
for observed oxidative stress in PKU could be a de- U. N. Antioxidant and thyroid hormone status in selenium-defi-
creased formation of ubiquinone 10 [5]. Ubiquinone cient phenylketonuric and hyperphenylalaninemic patients1,2.
Am. J. Clin. Nutr. 72:976 –981; 2000.
deficiency has been related to high Phe levels [34] that [8] Heales, S. J. R.; Bolaños, J. P.; Brand, M. P.; Clark, J. B.; Land,
may cause inhibition in the activity of 3-hydroxy-3- J. M. Mitochondrial damage: an important feature in a number of
methylglutaryl CoA reductase, a key enzyme for choles- inborn errors of metabolism? J. Inherit. Metab. Dis. 19:140 –142;
1996.
terol biosynthesis in mevalonate pathway. Same pathway [9] Bird, S.; Miller, N. J.; Colins, J. E.; Rice-Evans, C. A. Plasma
is also involved in synthesis of the poliprenyl side chain antioxidant capacity in two cases of tyrosinaemia type 1: one case
of ubiquinone. It was shown that both Tyr and ubiqui- treated with NTBC. J. Inherit. Metab. Dis. 18:123–126; 1995.
[10] Rottoli, A.; Lista, G.; Zecchini, G.; Butté, C.; Longhi, R. Plasma
none 10 levels were significantly decreased in PKU selenium levels in treated phenylketonuric patients. J. Inherit.
patients [5]. (v) Increased Phe was shown to inhibit the Metab. Dis. 8:127–128; 1985.
Na-K ATPase enzyme [35]. An inhibition of this mem- [11] Darling, G.; Mathias, P.; O’Regan, M.; Naughten, E. Serum
selenium levels in Individuals on PKU Diets. J. Inherit. Metab.
brane- bound enzyme in mitochondria may increase the Dis. 15:769 –773; 1992.
electron leak from the inner mitochondrial membrane, [12] Przyrembel, H.; Bremer, H. J. Nutrition, physical growth, and
thereby increasing free radical formation. (vi) High Phe bone density in treated phenylketonuria. Eur. J. Pediatr. 159:
S129 –S135; 2000.
concentrations inhibit amino acid transport systems; [13] Sarkissian, N. C.; Boulais, D. M.; McDonald, D. J.; Scriver, C. R.
therefore, amino acids involved in making antioxidant A heteroallelic mutant mouse model: a new orthologue for human
molecules and antioxidant enzymes cannot be made ef- hyperphenylalaninemia. Mol. Genet. Metab. 69:188 –194; 2000.
[14] McCaman, M. W.; Robins, E. Fluorimetric method for the deter-
ficiently. This may put cells in a low antioxidant mode. mination of phenylalanine in serum. J. Lab. Clin. Med. 59:885–
In conclusion, previous studies in PKU patients and 890; 1962.
the present study in animals suggest that the oxidative [15] Winter, R. A.; Zukowski, J.; Ercal, N.; Matthews, R. H.; Spitz,
D. R. Analysis of glutathione, glutathione disulfide, cysteine,
stress observed results from as yet unknown reasons. homocysteine, and other biological thiols by high-performance
Although PKU is not a rare disease and its biochemical liquid photography following derivatization by N-(1-pyrenyl)ma-
defect has been known for many years, the brain damage leimide. Anal. Biochem. 227:14 –21; 1995.
[16] Draper, H. H.; Squires, E. J.; Mahmoodi, H.; Wu, J.; Agarwal, S.;
seen in PKU is not completely understood. When pa- Hadley, M. A. Comparative evaluation of thiobarbituric acid
tients are given a very strict diet at a very early age methods for the determination of malondialdehyde in biological
(within 3 weeks of age), mental retardation is prevented. materials. Free Radic. Biol. Med. 15:353–363; 1993.
[17] Tietz, N. W. Textbook of clinical chemistry. Philadelphia: W.B.
Until the underlying biochemical mechanisms are found Saunders; 1986.
for the brain damage seen in PKU, our studies suggest [18] Aebi, H. Catalase in vitro. Methods Enzymol. 105:121–126; 1984.
 Phenylketonuria and oxidative stress 911

[19] Bradford, M. A rapid and sensitive method for the quantitation of suring the breakdown of hydrogen peroxide by catalase. J. Biol.
microgram quantities of protein utilizing the principle of protein- Chem. 195:133–140; 1952.
dye binding. Anal. Biochem. 72:248 –256; 1976. [31] McDonald, D.; Bode, V.; Dove, W.; Shedlovsky, A. Pahhph-5: a
[20] Kaufman, S. An evolution of the possible neurotoxicity of me- mouse mutant deficient in phenylalanine hydroxylase. Proc. Natl.
tabolites of phenylalanine. J. Pediatr. 114:895–900; 1989. Acad. Sci. USA 87:1965–1967; 1990.
[21] Nemethm, I.; Boda, O. The ratio of oxidized/reduced glutathione [32] Swaiman, K. K.; Wu, S. R. Phenylalanine and phenylacetate
as an index of oxidative stress in various experimental models of adversely affect developing mammalian brain neurons. Neurology
shock syndrome. Biomed. Biochim. Acta 48:53–57; 1989. 34:1246 –1250; 1984.
[22] Meister, A.; Anderson, M. E. Glutathione. Ann. Rev. Biochem. [33] Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and
52:711–760; 1983. medicine. Oxford: Clarendon Press; 1989.
[23] Srivasta, S. K.; Beutler, E. The transport of oxidized glutathione [34] Artuch, R.; Colone, C.; Vilaseca, M. A.; Sierra, C.; Cambra, F. J.;
from human erythrocytes. J. Biol. Chem. 244:9 –16; 1969. Lambruschini, N.; Campistol, J. Plasma phenylalanine is associ-
[24] Pietz, J.; Kreis, R.; Rupp, A.; Mayatepek, E.; Rating, D.; Boesch, ated with decreased serum ubiqiunone 10 concentrations in phe-
C.; Bremer, H. J. Large neutral amino acids block phenylalanine nylketonuria. J. Inherit. Metab. Dis. 24:359 –366; 2000.
transport into brain tissue in patients with phenylketonuria. [35] Dwivedy, A. K.; Shah, S. N. Effects of phenylalanine and its
deaminated metabolites on Na⫹, K⫹-ATPase activity in synap-
J. Clin. Invest. 103:1169 –1178; 1999.
tosomes from rat brain. Neurochem. Res. 7:717–725; 1982.
[25] Smith, C. B.; Kang, J. Cerebral protein synthesis in a genetic
mouse model of phenylketonuria. Proc. Natl. Acad. Sci. USA
97:11014 –11019; 2000. ABBREVIATIONS
[26] Halliwell, B.; Chirico, S. Lipid peroxidation: its mechanism,
measurement, and significance. Am. J. Clin. Nutr. 57(Suppl.): G6PD— glucose-6-phosphate dehydrogenase
715S–725S; 1993. GPx— glutathione peroxidase
[27] Gutteridge, J. A. Aspects to consider when detecting and mea-
suring lipid peroxidation. Free Radic. Res. Commun. 1:173–184; GSH— glutathione
1986. GSSG— glutathione disulfide
[28] Wilke, B. C.; Vidailhet, M.; Favier, A.; Guillemin, C.; Ducros, V.; MDA— malondialdehyde
Arnaud, J.; Richard, M. J. Selenium, glutathione peroxidase
(GSH-Px) and lipid peroxidation products before and after sele-
m-HPA— mild-hyperphenylalaninemia
nium supplementation. Chin. Chim. Acta 207:137–142; 1992. PAH— phenylalanine hydroxylase
[29] Brigelius, R. Regulation of glucose-6-phosphate dehydrogenase Pah— phenylalanine hydroxylase locus
under oxidative stress. In: Rotilio, G., ed. Superoxidize and su- PKU— phenylketonuria
peroxide dismutase in chemistry, biology and medicine. Amster-
dam: Elsevier; 1986:401– 403. RBCs— red blood cells
[30] Beers, R. F.; Sizer, I. W. A spectrophotometric method for mea- TBARS— thiobarbituric acid reactive substances