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2 
*
Department of Pediatrics, Washington University School of Medicine, St. Louis, MO;
Department of Paediatrics, College of Medicine, University of Malawi, Blantyre, Malawi;
**
Division of Metabolism, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO; and
Department of Medical Microbiology, University of Liverpool, Liverpool, UK
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were measured on admission, and at 24 and 48 h. The 16 children
who received egg white-tryptophan had lower rates of urea
appearance than those who received milk [57 ± 30 vs. 87 ± 36 µmol/(kg · h), mean ± SD, P
< 0.02]. No significant differences were found in the rates of
whole-body protein turnover or in the concentration of any of the
acute phase proteins or cytokines. The concentration of interleukin 6
was consistent with an appropriate proinflammatory response and
correlated directly with the concentrations of C-reactive protein
(r = 0.67, P < 0.01) and
1-antitrypsin (r = 0.40,
P < 0.05). The findings suggest that egg
white-tryptophan is associated with less amino acid oxidation in
kwashiorkor and acute infection than is milk.
KEY WORDS: • malnutrition • kwashiorkor • protein metabolism • acute phase response • stable isotopes • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Children with kwashiorkor have a higher incidence of
serious systemic infection than marasmic or well-nourished
children, and a higher case fatality rate (Freidland 1992
, McLaren et al. 1969
). Kwashiorkor is
associated with decreased rates of whole-body protein turnover,
which do not increase with the physiologic stress of acute infection as
they do in a well-nourished state (Manary et al. 1997a
). This suggests that endogenous amino acid release from
proteolysis is not as great in kwashiorkor, and amino acids may not be
as readily available for the synthesis of acute phase proteins. Thus,
the amino acid composition of the dietary protein may be more important
in facilitating the appropriate acute phase response to infection in
kwashiorkor. A dietary protein source composed of essential amino acids
that more closely resembles that of acute phase proteins may result in
less nitrogen wasting and a more vigorous acute phase response.
Previously, we tested this hypothesis by comparing milk and egg white
as dietary sources of protein and found the protein kinetic data
consistent with the hypothesis (Manary et al. 1997b
).
Milk is the dietary protein recommended for the initial treatment of
kwashiorkor, when infection is most likely to be concurrent
(Waterlow 1992
). Standard treatment recommendations call
for a low protein intake initially, 0.7 g/(kg · d). On the basis of
the amino acid composition of a typical mixture of acute phase proteins
(Reeds et al. 1994
), the fraction of each amino acid
provided by milk, egg white and egg white-tryptophan is shown in
Table 1
. Tryptophan is the limiting amino acid for both egg white and milk, and
the addition of a small amount of tryptophan (1 g/70 g, 1.5%) to egg
white gives it an amino acid composition more similar to the typical
acute phase response than milk. This study tested the hypothesis that
there would be less urea produced and higher concentrations of acute
phase proteins in children with kwashiorkor and an acute infection when
egg white-tryptophan is used as a dietary source of protein
compared with milk.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Feedings were administered in equal amounts per kilogram body weight
every 2 h; children unable to take feedings by mouth were fed
through a nasogastric tube. Children who were admitted during wk 1, 2
and 5 of the study were fed egg white-tryptophan, and those
admitted during wk 3 and 4 were fed milk. The children receiving egg
white as a protein source also received 10 mg/kg of tryptophan (Austen
Chemical, Buffalo Grove, IL) added to their feedings. Acute infection
was defined by a persistent fever, clinical signs of sepsis with a
positive blood or urine culture, clinical signs of falciparum malaria
with a positive smear for malaria parasites, dysentery, or cough and
tachypnea with a focal infiltrate on chest X-ray. The infections
were believed to be acute because, according to the caretaker’s
report, each child’s clinical condition had worsened within the day
before admission.
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). At 19 h
after admission, the
15N2-urea [prime, 21.5
µmol/kg; infusion, 2.15 µmol/(kg · h)]
and 1-13C-leucine [prime, 5.44
µmol/kg; oral administration, 5.44
µmol/(kg · h)] tracers were started and continued for
5–7 h. After 23.5 h (24 h after admission), a 1-mL blood sample
was drawn to measure isotopic abundance of urea and leucine.
Serum samples were analyzed by gas chromatography quadrapole mass
spectrometry after the urea was converted to its
t-butyldimethylsilyl derivative and the leucine was
converted to its heptafluorobutryl n-propyl ester
(Manary et al. 1997a
).
15N2-Urea isotope abundance
was determined with the use of electron impact ionization with selected
ion monitoring at m/z 231 and 233.
1-13C-Leucine isotope abundance was detected with
the use of positive chemical ionization with ion monitoring at
m/z 370 and 371.
The rates of appearance (Ra) of urea and leucine were
calculated from the following equation, derived from a simple mass
balance (Manary et al. 1997a
): Ra = [Ei/(Ep - 1)] x I where
Ei is the isotopic enrichment of the tracer infused
(98–99%), Ep is the isotopic enrichment of the tracer in
serum and I is the infusion rate of the tracer. The plasma
urea appearance rate was used to estimate the rate of amino acid
oxidation by assuming that the amino nitrogen from all oxidized leucine
is first incorporated into urea and that there is no nitrogen recycling
from urea. It was also assumed that the ratio of amino nitrogen from
leucine to total urea nitrogen is the same as the ratio of leucine to
total body nitrogen (3.817 mmol leucine/g N). Nitrogen recycling from
urea refers to the secretion of urea into the gastrointestinal tract,
followed by the breakdown of urea by bacterial ureases and the
reincorporation of this nitrogen into amino acids. It can be
quantitated by measuring the amount of
15N1 from
15N2-urea tracer that
appears in amino acids or urea. The plasma leucine appearance rate was
used to calculate the rates of whole-body protein synthesis and
breakdown, after accounting for the dietary leucine intake and the
estimated leucine oxidation (Manary et al. 1997b
). The
isotopic enrichment of leucine, rather than -ketoisocaproic acid,
was used because when leucine tracers are administered orally,
extracellular leucine enrichment best approximates the intracellular
isotopic leucine enrichment (Matthews et al. 1993
). The
care of the children, dietary management, metabolic studies and
calculations were the same as those for a previous study in which egg
white and milk (without additional tryptophan) were compared as dietary
sources of protein (Manary et al. 1997b
). The study was
approved by the Health Science Research Committee in Malawi, the Human
Studies Committee of Washington University and the Research Committee
of the Faculty of Medicine of the University of Liverpool.
The serum concentrations of seven acute phase proteins (C-reactive
protein, haptoglobin, 1-acid glycoprotein,
1-antitrypsin, C3, C4 and properidin factor B)
were measured in each child on admission, 24 and 48 h after
admission, using rate nephelometry (Beckmann Array 360; Beckmann, High
Wycombe, UK). The serum concentrations of interleukin 6 (IL-6) and
tumor necrosis factor- (TNF-) were determined at the same time
points with the use of ELISA kits that measure individual cytokine
concentrations with a panel of monoclonal antibodies (Medgenix,
Watford, United Kingdom). These assays detect total cytokine
concentration (both free and bound to soluble receptors) in the serum.
Comparison of the protein kinetic data between the egg white-tryptophan and the milk protein groups were made using Student’s t test. Comparison of the acute phase protein and cytokine data was made using nonparametric statistical methods, the Wilcoxon signed-rank test for continuous measures and Fisher’s exact test for dichotomous measures. Pearson’s coefficient of correlation was used to examine the relationship between acute phase protein and cytokine concentrations. A P-value < 0.05 was considered significant for all tests. Anthropometric Z-scores were calculated using Epi Info 6 software (Centers for Disease Control, Atlanta, GA) based on data from the National Center for Health Statistics (1979–1980).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); all eligible children participated in the study. There were no
significant demographic differences between the two dietary groups.
Three children receiving egg white-tryptophan and six receiving
milk died during their hospitalization (P > 0.20)
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Children receiving egg white-tryptophan had a lower plasma urea
appearance rate than those receiving milk protein (Fig. 1
). No differences were found in the rates of whole-body protein
synthesis and breakdown (Fig. 2
). The children receiving egg white-tryptophan used 98% of the
available leucine for new protein synthesis compared with 97% for
those receiving milk.
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There were no significant differences in the concentration of any of
the acute phase proteins between the two dietary groups (Table 4
). Similarly, the changes in acute phase protein concentrations from
admission to 24 and 48 h were not different between the two
groups. C-reactive protein and 1-acid
glycoprotein were the only acute phase protein concentrations that
increased above normal in more than half of the children (Table 5
). There were no significant differences in the concentrations of IL-6
or TNF- between the two dietary groups (Table 6
). The serum concentrations of C-reactive protein and
1-antitrypsin were directly correlated with
the concentration of IL-6 (Fig. 3
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The acute phase response has been observed with a variety of viral,
bacterial and parasitic infections (Gabay and Kushner 1999
). It is mediated by cytokines produced by endogenous
macrophages and monocytes. However, specific infections or infectious
agents might stimulate the production of cytokines to differing
degrees, and thus change the protein kinetic response. Because there
were very few children with any one specific infection in this study,
we were unable to draw any conclusions about how this might have
confounded the results. The limitations of the leucine and urea tracers
and the methods and calculations used have been discussed previously
(Manary et al. 1997a
and 1997b
). We assumed that plasma
tracer abundance had reached a steady state (for the purposes of the
metabolic calculations). There were no significant changes in the
isotopic enrichment of plasma leucine or urea after 4.5 h,
verifying the validity of the assumption. Oral leucine tracers were
used in this study because they are less invasive in critically ill
children, and the tracer amino acid is subject to the same metabolic
alterations as the dietary amino acids. Although there were no
differences in the serum concentrations of acute phase proteins between
the two dietary groups, the absolute synthesis rates of acute phase
proteins were not measured.
Very few studies of whole-body protein kinetics in children
< 5 y of age are available, primarily because very young
children cannot cooperate with the collection of expired air samples
for direct measurement of 13CO2 production
(Bodamer et al. 1997
). The paucity of data from this age
group has limited the utility of amino acid stable isotope tracer
techniques in children (Bodamer et al. 1997
). Our use of
the urea appearance rate to estimate amino acid oxidation is
unconventional, but it is a method by which young children can be
studied successfully. We believe that little urea recycling was
occurring during this short study because there was no isotopic
enrichment seen in the (m + 1) ion of urea. All of the assumptions made
in the calculations were applied equally to both groups of children,
and therefore any inaccuracies introduced by the use of an erroneous
factor or fraction would not affect the tests of statistical
significance between the two groups.
As in previous work, we found that the acute phase response in children
with kwashiorkor and acute infection was blunted (Doherty et al. 1993
, Hafez et al. 1977
, Olusi et al. 1976
, Razban et al. 1975
); only C-reactive
protein and 1-acid glycoprotein were elevated in more
than half of the children. In well-nourished children with acute
infection, the concentration of each of the acute phase proteins
(except C4) increases several fold (Dowton and Colten 1988
). These plasma proteins are part of an appropriate
immune response and are synthesized in quantities up to 1.2
g/(kg · d) (Waterlow 1991
). The observed
concentrations of IL-6 and TNF- in these 30 children are similar to
those reported in well-nourished patients with serious infection
and septic shock (Puren et al. 1995
, Sullivan et al. 1992
). In this study, the plasma concentration of IL-6 was
correlated directly with C-reactive protein and
1-antitrypsin concentrations. In vitro work suggests
that the response of IL-6 to infection may be blunted in
protein-energy malnutrition, but more recent clinical work
demonstrated that IL-6 concentrations were elevated in kwashiorkor and
infection (Doherty et al. 1994
, Sauerwein et al. 1997
). The concentrations of IL-6 observed here are consistent
with an appropriate proinflammatory cytokine response. Therefore, we
speculate that if more amino acids were available, more acute phase
proteins would have been synthesized.
Children in this study received 1.2 g/(kg · d) of dietary protein,
with a protein to energy ratio of 6.9%. Assuming that 0.6
g/(kg · d) was required to replace obligatory nitrogen losses
through skin, urine and feces (Waterlow 1986
), only 0.6
g/(kg · d) was available for the synthesis of acute phase proteins
from exogenous amino acids. If 77% of these amino acids could be used
(84% for egg white-tryptophan, 71% for milk), only 0.45
g/(kg · d) of acute phase proteins could be synthesized from
dietary amino acids. Whole-body protein turnover studies have found
that a vigorous acute phase response is associated with an increase in
whole-body protein synthesis of 0.6–0.8 g/(kg · d)
(Cayol et al. 1995
, Fong et al. 1994
),
and the magnitude of the acute phase response has been estimated to be
as high as 1.2 g/(kg · d) (Waterlow 1991
). The
availability of endogenous amino acids is markedly restricted in severe
malnutrition in an effort to conserve amino acids and energy
(Waterlow 1992
). Standard therapeutic recommendations in
the initial treatment of kwashiorkor call for a low energy, low protein
diet (Waterlow 1992
). These recommendations are based on
the clinical experience from the best tropical metabolic units 30 years
ago and continue to be corroborated by current work (Collins et al. 1998
). Although it appears that greater dietary protein
intakes may be required to provide sufficient amino acids for an
appropriate acute phase response in kwashiorkor, increases in the
protein to energy ratio above 8.5% are contraindicated.
Combinations of other cereals and legumes (corn, rice, beans, wheat,
sorghum and soybean) as protein sources provide a lesser amount of the
total amino acids required for the synthesis of acute phase proteins
than does egg white-tryptophan (Pennington 1994
). On
the basis of this study, we recommend a trial of 1.5 g/(kg · d) of
egg white-tryptophan as a dietary source of protein in the initial
treatment of kwashiorkor (protein to energy ratio of 8.5%) to
determine whether the increased nitrogen conservation with the egg
white-tryptophan diet is associated with any clinical benefits.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: HIV, human immunodeficiency
virus; IL-6, interleukin-6; TNF-, tumor necrosis factor-.
Manuscript received August 10, 1999. Initial review completed September 13, 1999. Revision accepted October 19, 1999.
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