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Q J Med 1999; 92:495–503
Original papers
QJM
Hyperphenylalaninaemia in children with falciparum malaria
C.O. ENWONWU1,2 B.M. AFOLABI3, L.A. SALAKO3, E.O. IDIGBE3,
H. AL-HASSAN4 and R.A. RABIU5
From the Departments of 1OCBS, School of Dentistry, and 2Biochemistry, School of Medicine,
University of Maryland, Baltimore, Maryland, USA, 3Nigerian Institute of Medical Research,
Lagos, 4Specialist Hospital, Sokoto, and 5Massey Street Children Hospital, Lagos, Nigeria
Received 5 February 1999 and in revised form 5 July 1999
Summary
Brain monoamine levels may underlie aspects of the
cerebral component of falciparum malaria. Since
circulating amino acids are the precursors for brain
monoamine synthesis, we measured them in malaria
patients and controls. Malaria elicited significantly
elevated plasma levels of phenylalanine, particularly
in comatose patients, with the Tyr/Phe (%) ratio
reduced from 83.3 in controls to 39.5 in infected
children, suggesting an impaired phenylalanine
hydroxylase enzyme system in malaria infection.
Malaria significantly increased the apparent K for
m
Trp, Tyr and His, with no effect on K
for Phe.
m(app)
Using the kinetic parameters of NAA transport at
the human blood–brain barrier, malaria significantly
altered brain uptake of Phe (+96%), Trp (−28%)
and His (+31%), with no effect on Tyr (−8%),
compared with control findings. Our data suggest
impaired cerebral synthesis of serotonin, dopamine
and norepinephrine, and enhanced production of
histamine, in children with severe falciparum
malaria.
Introduction
Malaria is a major cause of morbidity and mortality
in sub-Saharan Africa, and kills more than one
million children annually.1,2 The factors that determine whether a child develops mild or severe malaria
are numerous and complex.1 Severe Plasmodium
falciparum malaria is a multisystem disease involving
several metabolic disturbances and endocrine dysfunctions,1,2 with recurrent paroxysms of high fever,
persistent vomiting, confusion, seizures, and loss of
consciousness (cerebral malaria) among the danger
signs.2–5 Resting energy expenditure may be
increased by as much as 30%.3 The fever elicits
several host responses, such as altered fat and
carbohydrate metabolism, increased catabolism of
skeletal muscle, complex alterations in the metabol-
ism of individual amino acids, and enhanced nitrogen
excretion, with changes in the priority for protein
synthesis, and increased gluconeogenesis, among
others.2,6,7 The host’s responses, especially those
mediated through cytokine cascade, may determine
disease severity.8,9 The cytokines stimulate release of
glucocorticoids which in turn inhibit cytokine gene
expression, in addition to promoting catabolism of
muscle proteins and increased mobilization of amino
acids to the liver.9–11 Abnormalities of hepatic2,12
and renal13 functions are seen in a significant proportion of children with severe malaria.
Plasmodium falciparum infection is considered
one of the commonest causes of acute encephalopathies in children residing in falciparum-malaria-
Address correspondence to Professor C.O. Enwonwu, Departments of Biochemistry and OCBS, Schools of Medicine and
Dentistry, 666 West Baltimore Street, Room 4G31, Baltimore, MD 21201-1586, USA
© Association of Physicians 1999
496
C.O. Enwonwu et al.
infected countries.2,14 There is good evidence that
some common disorders of central monoamine metabolism may underlie the pathogenesis of metabolic
encephalopathies.15,16 Studies in mice and rats with
Plasmodium berghei infection indicate significantly
reduced whole brain contents of 5-hydroxytryptamine (serotonin) and norepinephrine, while
levels of dopamine and histamine remain unaltered.17
Large neutral amino acids circulating in blood plasma
are precursors for the synthesis in brain of the
monoamines, as well as other putative neurotransmitters such as carnosine, and important cofactors such
as S-adenosyl-methionine,18 and alterations in their
concentrations or turnover in the brain could affect
monoaminergic functions.19 This latter idea derives
from reports that the rate-limiting steps in cerebral
biosyntheses of these neurotransmitters are not saturated by normal concentrations of the relevant precursor amino acids in brain cells, and that, therefore,
amino acid transport at the blood–brain barrier (BBB)
plays a critical role in the overall regulation.18
Studies of changes in the free amino acid (AA)
plasma pool in malaria are limited, contradictory,
and based mainly on the avian malaria Plasmodium
lophurae in ducklings,20,21 with the animals demonstrating selective increases in plasma concentrations
of specific amino acids.21 Since the brain is uniquely
vulnerable to plasma hyperaminoacidaemia,18 our
study was designed to examine the changes in
plasma free amino acids in African children with
P. falciparum malaria. We also evaluated plasma
levels of free cortisol and ascorbate, as additional
indices of the metabolic consequences of the disease.
Methods
Approval for this study was by the Ethical Committees
of the Nigerian Institute of Medical Research, Yaba,
Lagos, the Lagos State Health Management Board,
and the Ministry of Health, Sokoto State. Informed
consent was obtained from the accompanying parents or guardians of the patients, who were assured
of the confidentiality of individual results.
Study areas and patients
The studies were carried out at the Massey Street
Children’s Hospital, Lagos, and the Sokoto Specialist
Hospital, Sokoto City, Sokoto State. Both hospitals
are located in very high-density areas with deplorable
environmental sanitation, and grossly inadequate
basic amenities such as potable water as well as an
unreliable electricity supply. The former hospital
draws its patients from a population of about 2
million people residing in the Lagos metropolis,
while Sokoto Specialist Hospital draws its patients
not only from Sokoto State, but also from the two
neighbouring states of Kebbi and Zamfara. As in
most states in Nigeria, malaria is endemic in the
study areas, especially during the rainy season, with
Plasmodium falciparum predominating.
We recruited 28 randomly-selected children (13
females and 15 males, ages 14–156 months,
mean±SD age 65.14±49.05) from the lowsocioeconomic urban communities in Sokoto and
Lagos. These children, 13 from Sokoto and the rest
from Lagos, were selected from a much larger group
of patients who presented at the hospitals during a
3-week period in 1997, with fever of varying degrees
ranging from 38.0 to 40.8 °C. The children recruited
from the two hospitals showed no significant difference in mean weights for age Z-scores and were,
therefore, treated as one single group for the purposes
of the study. Most of the children complained of
persistent fever of a few days duration, with loss of
appetite, nausea, vomiting and headache. Exclusion
criteria were presence of a chronic illness, congenital
abnormalities, other overt infectious diseases, severe
malnutrition as exemplified clinically by kwashiorkor
or marasmus, and food consumption within 6 h prior
to presentation at the hospital. Each patient was
given a full clinical examination. In particular, presence of jaundice, pallor, hepatomegaly and spleen
enlargement were carefully documented. Four of the
patients presented at the hospital with severelyimpaired consciousness, absent corneal reflexes,
repeated convulsions of the whole body, poor motor
response to noxious stimuli, evidence of respiratory
distress, and other features associated with cerebral
malaria.22 The depth of coma was not measured. For
comparative purposes, 28 children (mean age
72.1±18.8 months), distributed equally between
genders and without malaria and overt signs of other
infections, were recruited as the control group. These
control children (14 each from Lagos and Sokoto)
resided in the same low-socioeconomic-status urban
communities as the children with malaria. They were
enrolled in the study when they visited the hospitals
(out-patient clinics) and the primary health-care
centres for routine immunizations and a countrywide
oral health screening program. The control children
were recruited into the study during the same time
interval as the malaria-infected children, and like the
latter, they were predominantly of Hausa, Fulani,
and Yoruba ethnic origins.
Clinical methods
Axillary temperature was measured with a mercury
thermometer, using 1 min stabilization time. Pyrexia
was defined as any temperature of 37.5 °C and
above. Finger prick blood was obtained from each
malaria patient for examination for malaria parasites
497
Hyperphenylalaninaemia in malaria
and determination of packed cell volume (PCV).
Thick and thin blood films were prepared on the
same slide for each child. The slide was stained
using buffered Giemsa23, and examined under oil
immersion at ×700 magnification. Laboratory diagnosis of malaria was based on observation of any
stage of development of malaria parasites within the
red blood cells. Species of the parasite was
determined from the thin film. Parasite density was
determined as previously described,23 using the thick
film preparation. Four hundred fields were examined
and certified to contain no parasites before a slide
was declared negative.
Anthropometry
Body weight of each child was measured to the
nearest 0.1 kg using a scale whose precision was
frequently checked. Height was measured to the
nearest mm. Assessment of nutritional status was
predicated on weight-for-height as an index of current nutritional state, and on height-for-age (stunting)
as an index of past nutrition. Using computer programs developed by the WHO, Geneva, and the
CDC, Atlanta, for nutritional anthropometry (Epi Info
version 5.0 lb, 1993), weight-for-age, height-for-age,
and weight-for-height Z-scores were calculated, and
interpreted relative to the standard values of the
National Center for Health Statistics (NCHS) published by the US Department of Health, Education
and Welfare.24 The Z-score cutoff point chosen was
−2 standard deviations from the reference median.
Biochemical studies
Sample collection
Venous blood was collected at 09:00–11:00 h into
heparinized tubes following fasting periods that lasted
6–12 h for the various subjects. At this stage, one
male patient with malaria was excluded from the
group because of inadequate blood collection. The
blood-filled vacutainer tubes were retained in an
ice-cooled chest until centrifuged (2000 g for 10 min)
usually within 30 min after collection. The separated
plasma was divided into aliquots for storage at
−70 °C until analyzed. The aliquot for cortisol assay
was stored in a siliconized tube. The aliquot for
ascorbate assay was stabilized with an equal volume
of freshly prepared 10% metaphosphoric acid
before storage.
Assays
Levels of free amino acids in plasma were measured
by reverse-phase HPLC following pre-column derivatization with phenylisothiocyanate.25 Phenylalanine
shares the same transporter at the blood brain barrier
(BBB) with valine, methionine, isoleucine, leucine,
tyrosine, tryptophane and histidine, and all these
amino acids possess different affinities for the transport site.26 To calculate brain uptake of these amino
acids, the following equations26,27 were used:
A
B
[AA]
K
=K 1+S
m(app)
m
K (AA)
m
[AA].v
max
=
+[AA]. K
d
[AA]+K
m(app)
where K
is the apparent or normalized Km for
m(app)
amino acid (e.g. Phe), K is the measured K for
m
m
that amino acid, [AA]/K (AA) is the concentration
m
divided by the measured Km for all other amino
acids which share that transporter (e.g. Tyr, Trp, Ile,
Leu, Met, Val, His), v is brain uptake for that amino
acid, v is the measured v for that amino acid,
max
max
[AA] is the plasma concentration of that amino acid,
and K is the measured passive diffusion constant.
d
Values for K , v , and K for the large neutral
m max
d
amino acids (LNAA) at human brain capillaries have
been reported by Hargreaves and Pardridge.27
Determination of cortisol concentration in plasma
was carried out with the ‘Gamma Coat’ 125IRadioimmunoassay Kit (Incstar), as previously
described.28 The kit contained test tubes coated with
rabbit anti-cortisol serum, 125I-labelled cortisol
in phosphate-buffered saline, ANS (8-anilino1-naphthalene sulphonic acid) with 0.02 M sodium
azide preservative, cortisol serum blank (cortisol-free
processed human serum), phosphate-buffered saline,
and cortisol standards in processed human serum in
concentrations of 0, 1, 3, 10, 25 and 60 mg/dl (0,
28, 83, 276, 690 and 1655 nmol/l). Using unextracted plasma (10 ml), cortisol concentrations were
determined as indicated in the instruction manual
that came with the kit. Precision was checked using
recovery from spiked, pooled serum samples, and
was in the range of 93–102%. The reference ranges
for plasma cortisol levels were 193–690 nmol/l
(mornings) and 55–248 nmol/l (evenings).
Plasma concentration of total ascorbic acid (dihydro-plus-dehydro-forms) was determined following
derivatization with 2,4-dinitrophenylhydrazine as
previously described.29 For interpretation of the data,
we considered values <11 mmol/l to signify frank
deficiency, values between 11 and 23 mmol/l as
marginal or moderate risk of developing clinical deficiency signs, and values >23 mmol/l as
normal.30
Statistical analysis
Results were expressed as means±SD. Statistical
differences between means were evaluated by using
Student’s t-test when comparing groups, or by an
analysis of variance of repeated measurements when
498
C.O. Enwonwu et al.
comparing more than two situations. Differences
between proportions were analyzed using x2 test.
The level of significance was chosen as p<0.05.
Results
With the exception of four children who were
comatose, the rest were alert on admission. The
main presenting symptoms in the 27 patients
included loss of appetite (96%), vomiting (70%),
convulsions (30%), headache (22%), diarrhoea
(26%), fever (100%), splenomegaly (10%) and hepatomegaly (24%). Pre-medication with analgesics
(62%), chloroquine (41%), antihistaminics (11%), or
multivitamins including folate (33%) was noted in
some of the patients. The packed cell volume (PCV)
in the sick children varied from 15% to 51%, with
23% of them having a PCV of less than 30%.
Similarly, marked variability was noted in malaria
parasite density among the patients (range
22–386 076/ml, with geometric mean parasite density
of 2669/ml). The percentages of the patients who
were underweight, stunted or wasted, as indicated
by their weight-for-age (WAZ), height-for-age (HAZ),
and weight-for-height (WHZ) Z-scores were 30.8,
17.4, and 20.0, respectively, an observation suggestive of protein-energy malnutrition (PEM) in a good
number of the children.31
The most prominent changes in the plasma free
amino acid profiles in the children with malaria in
comparison to the non-infected children were significant ( p<0.05) increases in phenylalanine and
histidine, and a decrease in arginine levels (Table 1).
Non-significant alterations between the two groups
were observed in the levels of most of the dietary
essential and non-essential amino acids. The ratio
(%) of Tyr/Phe decreased markedly from 83.3 in the
non-infected children to 39.5 in the malaria group.
The sum (SNAA) of the large neutral amino acids,
including histidine (Val, Met, Ile, Leu, Phe, Trp, Tyr,
His) which share the same transporter at the BBB,
increased by 32% in the malaria group compared
with the level in the non-infected group.
Phenylalanine accounted for a good proportion of
the increase in the children with malaria. For initial
semi-quantitative evaluation of the potential effects
of changing plasma amino acid concentrations on
brain availability of each individual large neutral
amino acid, we calculated the amino acid ratio,32
an approach that incorrectly assumed that all the
large neutral amino acids have the same affinity for
the BBB transporter.18,26,27 Nonetheless, the crude
assessment indicated significantly increased Phe ratio
(Phe/SNAA)% from 12.2 in the non-infected group
to 24.7 in the malaria group (Table 1). The His and
Trp ratios were significantly altered in the malaria
group, but the Tyr ratio was unaffected. Using the
K (apparent) for each amino acid,26,27 and published
m
kinetic parameters of transport (K , V and K ) for
m max
d
the NAA at the human BBB,27 we derived more
accurate data on the brain uptake of the various
amino acids. As shown in Table 2, the apparent K
m
for Phe was not altered in the malaria group. In
contrast, the infection produced a 25% significant
increase in apparent K in each of Trp, Tyr, and His
m
compared with values for the control children.
Malaria infection elicited significant increases in
brain uptake of Phe (+96%) and His (+31%), a
reduction in uptake of Trp (−28%), and no change
in Tyr uptake, compared with findings in the control
group (Table 2). The patients with malaria were
further classified into three sub-groups of those who
were comatose, those with parasite density in excess
of 13 000/ml, and patients pre-medicated with folate
before presentation at the study site. Excluded from
the second sub-group were children in coma or
those pre-medicated with folate. As shown in Table 3,
the four patients in coma showed the highest degree
of hyperphenylalaninaemia, which was not markedly
different from the level in patients with high parasite
density, but significantly higher (+86%) than the
level observed in nine children with a history of premedication with folate.
As summarized in Table 4, the malaria patients
demonstrated prominently increased plasma free
cortisol concentrations, with marked reductions in
mean plasma total ascorbate levels compared to
findings in the non-infected control children. Only
4 of the 27 patients (15%) had plasma ascorbate
level within the normal range, while the rest had
levels <23 mmol/l.
Discussion
Plasmodium falciparum is considered one of the
commonest causes of acute encephalopathies in
children in most endemic countries.1,4,14 Since
disorders of central monoamine metabolism may
underlie the pathogenesis of metabolic encephalopathies,16,18 the present studies focused primarily on
circulating plasma levels of the large neutral amino
acids, many of which serve as precursors for synthesis
in brain of the monoamines (serotonin, dopamine,
noradrenaline, and histamine).18,19,26,33 As shown in
Table 1, increased plasma Phe was the most conspicuous change in the children with malaria, with
Phe levels 2–3-fold those seen in uninfected children,
and within the range of values reportedly characteristic of some individuals heterozygous for phenylketonuria.34 Our findings were consistent with reports
showing plasma Phe concentrations 45–86% above
499
Hyperphenylalaninaemia in malaria
Table 1 Plasma amino acid levels (mmol/l)
Amino acids
Malaria group
(n=24)*
Controls
(n=26)*
Threonine
Valine
Methionine
Isoleucine
Leucine
Phenylalanine
Tryptophane
Lysine
Histidine
Arginine
Tyrosine
Glutamic acid
Serine
Asparagine
Glycine
Taurine
Alanine
SNAA
Tyr/Phe(%)
Phe/SNAA (%)
Trp/(SNAA (%))
Tyr/SNAA (%)
His/SNAA (%)
SEAA
SNEA
SEAA/SNEA (%)
91.46±29.96
200.52±55.70
27.56±7.46
50.60±9.05
140.48±45.50
155.11±23.79a
38.90±7.11
156.46±42.11
108.77±20.26a
85.25±23.43a
61.30±14.86
268.28±90.30
171.98±53.18
58.47±9.52
257.99±78.35
116.27±39.63
378.53±60.09
783.24
39.52
24.69
5.23
8.49
16.13
1055.11
1312.82
80.37
102.11±18.20
188.08±26.55
25.13±5.65
52.62±11.05
96.55±21.77
64.52±8.08**
44.30±10.11
118.68±22.14
68.79±12.99**
123.10±18.77**
53.72±11.18
188.62±25.56
165.30±25.57
69.82±11.88
331.67±69.97
146.13±44.21
390.27±41.77
593.71
83.26**
12.19**
8.06**
9.95
13.10**
883.88
1345.53
65.69
Data are means±SD. SNAA, sum of large neutral amino acids (Val, Met, Ile, Leu, Phe, Trp, Tyr, His); SEAA, sum of
essential amino acids (includes Arg, His); SNEA, sum of non-essential amino acids. * Three and two haemolysed samples
discarded from malaria and control groups, respectively. ** Significantly different ( p<0.005 or 0.05) from same entry in
other group.
Table 2 Apparent K and calculated brain uptake of amino acids
m
Amino acid
Phenylalanine
Tryptophan
Tyrosine
Histidine
Apparent K (mM)
m
Uptake (v) (pmol/min)
Controls
Malaria group
Controls
Malaria group
0.34±0.03
4.04±0.12
1.71±0.06
6.86±0.11
0.35±0.08
5.03±0.07
2.13±0.02
8.50±0.14
1.02
0.20
0.36
0.43
2.00
0.14
0.33
0.56
(+2.3%)
(+24.5%)*
(+24.7%)*
(+23.9%)*
(+96.1%)*
(−28.2%)*
(−8.3%)
(+30.7%)*
Data (means±SD) based on findings in 24 children in each group of children. Values in parentheses represent percent
change from control value. * Significantly different (p<0.05) from corresponding control value.
normal, in ducklings infected with Plasmodium
lophurae.20,21
Factors predisposing to hyperphenylalaninemia
include glucocorticoid-mediated catabolism of skeletal muscles in infections with increased release of
Phe, PKU heterozygosity, hepatic disease and renal
pathology with release of putative uraemic toxins
that may affect phenylalanine hydroxylase, among
others.7,18,35 Some of these factors, particularly
impaired liver function2,36 and hypercortisolaemia,10
as indicated in Table 4, are frequent features of
severe falciparum malaria in children. The pathological consequences of hyperphenylalaninemia are
mainly in the brain,37,38 and may include such
features as mental retardation, spasticity, and seizures.18 Indeed, a tripling of plasma Phe level to
about 200 mM, as observed in the present study
(Tables 1 and 3), may cause seizures, mood changes,
insomnia, nausea, abdominal pain and diarrhoea,18,26
which are among the features commonly
500
C.O. Enwonwu et al.
Table 3 Plasma phenylalanine and tyrosine in subgroups of malaria patients
Subgroup
Amino acid (mmol/l)
Comatose patients (n=4)
Parasite density >13 000 per ml (n=6)**
Folate-treated (n=9)***
Phenylalanine
Tyrosine
Tyr/Phe (%)
241.93±46.34*
201.65±55.78
129.93±41.08*
77.88±26.78*
78.67±29.75
54.90±14.55*
32.3*
39.0
42.3*
* Subgroups significantly different from each other ( p<0.05). ** Comatose children as well as those treated with folate
were excluded from this sub-group. *** Sub-group did not include any comatose child.
Table 4 Plasma cortisol and ascorbate levels
Plasma cortisol (nM/l)
Ascorbate (mmol/l)
Malaria group
(n=24)
Non-infected controls
(n=24)
1021.26±351.94* (range 304–1518)
19.52±5.77*
502.18±182.55* (range 93–799)
26.44±3.42*
Data are means±SD. * Significantly different for malaria patients vs. controls.
encountered in severe malaria.2,4,14 In the very limited number of comatose patients evaluated in the
present study, mean plasma Phe level was as high
as 242 mmol/l, a value four-fold the normal Phe
concentration in plasma (Table 3).
Plasma amino acid abnormalities characterize several health conditions including protein energy malnutrition (PEM),39,40 infections,7,28,41,42 severe
trauma,43 and stress, as well as sepsis.44,45 In PEM,
with the possible exceptions of His and Phe, whose
plasma concentrations are marginally well maintained, all indispensable amino acids are reduced in
concentration so that EAA/NEA ratio declines, but
His/NEA ratio is slightly elevated.39,40,46 The activities
of key Phe39,47 and His46,47 catabolic enzymes are
reduced in PEM. In a study of stressed and/or septic
patients, Vente and colleagues44 noted a 70%
increase in plasma Phe over control level, with
virtually all the other essential amino acids in plasma
reduced by 10–30%, an observation prompting the
suggestion that a correlation exists between plasma
Phe levels and mortality during sepsis.44,45 There are
also reports that in experimentally-induced sand-flyfever virus infection in healthy human adults, plasma
amino acid levels compared with pre-inoculation
control levels were Phe (+4%), Val (−33%), Thr
(−26%), Leu (−33%), Ile (−31%), Tyr (−24%),
Trp (−15%), Met (−16%) and His (−17%).42 In
multiple-trauma patients compared with controls,
plasma Phe is elevated (+33%) whilst its urinary
excretion is increased by 13.3 times, mainly due to
a faster clearance rate.48 What emerges from the
numerous published studies on the alterations of
plasma amino acid levels in infections, sepsis, and
malnutrition is that: (i) not all individual essential
amino acids change in exactly the same manner;
(ii) the magnitude of the changes often correlates
with the degree of the host’s febrile response; and
(iii) the reported increases in plasma Phe in the
various conditions do not approach the magnitude
encountered in patients with severe malaria (Tables
1 and 3). It must also be emphasized that the critical
factors underlying brain uptake of any individual
amino acid under various conditions are not only
the plasma level of the specific amino acid, but also
the alterations in plasma concentrations of the amino
acids that share the same transporter with the former
at the BBB.18,26 The complex alterations in metabolism of individual amino acids in an infection are
influenced by fever.7 The African child suffering from
an acute episode of malaria may experience a resting
energy expenditure increase of about 30%.3
The phenylalanine hydroxylating system complex
consists of phenylalanine hydroxylase (PAH; EC
1.14.16.1), a regulatory cofactor tetrahydrobiopterin
(THB), and dihydropteridine reductase (DHPR; EC
1.6.99.7) which keeps the cofactor in its active
tetrahydro-form.49 DHPR deficiency and/or defective
synthesis of THB, will elicit inadequate THB-dependent hydroxylase activities (Phe hydroxylase, Tyr
hydroxylase, Trp hydroxylase), and consequently,
impaired synthesis of dopamine, norepinephrine and
serotonin, in addition to hyperphenylalaninaemia.49
In our study, patients pre-medicated with folic acid
showed less prominent hyperphenylalaninaemia
(Table 3), an observation suggestive of an increased
dietary requirement for this vitamin in falciparum
malaria and consistent with other reports of folate
deficiency in severe malaria.50 Plasmodium falciparum possesses an endogenous folate synthesis
Hyperphenylalaninaemia in malaria
pathway, but also utilises exogenous folate.51 Studies
of intraerythrocytic P. falciparum grown in continuous culture51 have shown that the parasite utilises
pterin from exogenous folate degradation as a precursor for synthesis of 5-CH3-H4 Pte Glu5 (5-methyltetrahydrofolate), and that the extremely low rate of
de novo synthesis from GTP or guanosine
(30.7±9.2 pmol/0.1 ml packed cells/24 h) as compared with the rate of synthesis from exogenous
folate, intact or degraded forms (1732±
207 pmol/0.1 ml packed cells/24 h) suggests a predominance of the latter pathway in the acquisition
of folate cofactors for the parasites. We have no
explanation for the observation that pre-treatment
with folic acid reduced the changes in plasma
phenylalanine and tyrosine levels in patients with
malaria (Table 3). It is possible that severe malaria
elicits deficient activity of DHPR through mechanisms that are still unclear. There are, however,
suggestions that DHPR may be involved in other
enzyme systems,52 and this enzyme may keep folate
in the active, tetrahydro form.53 The latter role could
explain the low serum folate levels observed in some
PKU patients with DHPR deficiency,54 and the inclusion of tetrahydrofolate in the therapy for these
patients.52,55
Phenylalanine is transported solely by the
L-system,18,27 and its K at the BBB is 4- to 29-fold
m
lower than those for other large neutral amino acids
which share the same transporter.27 As indicated in
Table 2, malaria infection had no effect on the
apparent K for Phe but elicited a significant increase
m
(+25%) in the apparent K for Trp, Tyr and His.
m
Consequently, the uptake of Phe into the brain was
calculated to be double in malaria patients compared
with the controls, while the uptake of Trp was
significantly reduced (Table 2). Both tyrosine
hydroxylase and tryptophane hydroxylase, ratelimiting enzymes in the syntheses of dopamine/
norepinephrine and serotonin, respectively, are competitively inhibited by increased phenylalanine.57
Loo58 has demonstrated serotonin deficiency in
experimental hyperphenylalaninaemia. Similarly, in
autopsied brains of PKU patients, Phe levels increase
five-fold, Tyr and Trp levels are reduced, and brain
contents of serotonin, dopamine and norepinephrine
substantially reduced.59 Some workers17 have argued
that the significantly reduced brain serotonin and
norepinephrine levels in rodents infected with
P. berghei may play a role in the occurrence of
cerebral vasodilation in malaria. The occurrence of
seizures in severe malaria2,4,5 may be related to
decreased brain levels of serotonin and catecholamines (putative inhibitory neurotransmitters)60 resulting
from hyperphenylalaninaemia-induced reduction in
brain levels of tryptophan and tyrosine. In an animal
model system of spontaneous seizures, brain levels
501
of the inhibitory monoamine neurotransmitters are
significantly decreased.61
The markedly increased brain uptake of histidine
in children with malaria (Table 2) would favour an
elevated brain level of histamine.62 The K for
m
L-histidine carboxylase (EC 4.1.1.22) in the brain is
much higher than the level that can be saturated by
normal brain contents of free histidine.62 Thus, elevated brain levels of histidine would promote
enhanced synthesis of histamine (imidazolethylamine).62,63 It is purely speculative at this stage whether
or not the brain burden of histamine is increased in
malaria, but this merits some careful studies, since
elevated brain levels of this monoamine may promote
increased cerebral capillary permeability, resulting
in cerebral oedema and elevated intracranial pressure, among other effects.63 These are pathophysiological alterations associated with cerebral malaria.2,4
Nitric oxide (NO) is implicated in some aspects
of the pathogenesis of severe malaria,64,65 and the
significantly reduced plasma arginine observed in
the malaria patients (Table 1) could be due to
increased utilization of this amino acid by the nitric
oxide synthase (NOS) pathway. Our studies also
demonstrated a two-fold elevation in plasma level
of free cortisol in the children with malaria compared
with the non-infected group (Table 4), an observation
consistent with reports by others10 and attributable
to stimulation of the hypothalamus-pituitary-adrenal
axis by pro-inflammatory cytokines.8–10 The increased
circulating levels of glucocorticoids would play a
role not only in mobilizing free amino acids from
the periphery to the liver, but also in suppressing
excessive/inappropriate cytokine production.7,9 The
marked reduction of plasma ascorbate in malaria
(Table 4) confirmed earlier reports by others,66 and
could be due to reduced dietary intake, and
enhanced utilization in cell mediated immunity as
well as in free radical quenching.
There are suggestions that there is a cerebral
component in virtually all cases of falciparum malaria.14 Despite the very limited number of children
with falciparum malaria examined in the present
studies, the findings suggest an urgent need to
evaluate some of the symptoms encountered in this
disease within the context of major alterations in the
metabolism of monoamines in the brain. It is perhaps
relevant that the consumer-initiated complaints associated with excessive intake of aspartame (L-aspartylL-phenylalanine methyl ester)-containing food products include mood changes, insomnia, seizures,
nausea, diarrhoea, abdominal pain, and irregular
menses.67 Some of these are features of severe
malaria.1,2,4,5 Unlike blood levels of aspartic acid,
blood concentrations of phenylalanine rise markedly
after ingestion of aspartame, and the rise is linearly
related to the dose of aspartame.68 The absence of
502
C.O. Enwonwu et al.
any substantial increase in plasma aspartic acid level
precludes this amino acid from participating in the
observed changes.60
17.
Acknowledgements
18.
This study was supported in part by the Nestle
Foundation, Lausanne, Switzerland. The excellent
administrative assistance of Ms. Judy Pennington is
also acknowledged.
19.
20.
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