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Health Canada, Nutrition Research Division, Bureau of Nutritional Sciences, Health Protection Branch, Banting Research Centre (AL: 2203 C), Tunney's Pasture, Ottawa, Ontario, Canada K1A OL2
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • infant formulas • tryptophan bioavailability • rat growth • brain serotonin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Sarwar et al. 1996
). Tryptophan
fortification of an adapted cow's milk-based infant formula was
reported to increase plasma tryptophan concentration to levels not
different from those found in breast-fed infants
(Fazzolari-Nesci et al. 1992
, Hanning et al. 1992
). However, 54% more tryptophan than that present in
breast milk had to be fed to barely increase the plasma concentration
to that found in breast-fed infants (Fazzolari-Nesci et al. 1992
). This would suggest a lower bioavailability of tryptophan
in infant formulas and/or higher susceptibility of crystalline
tryptophan to heat treatment than the bound tryptophan in the formula
proteins. Data on the bioavailability of tryptophan in infant formulas
are limited.
A change in the dietary supply of tryptophan may have both metabolic
and behavioral effects in infants (Steinberg et al. 1992
). For example, feeding infant formulas containing
different amounts of supplemental tryptophan to new-born infants
produced differences in sleep latency (Steinberg et al. 1992
). An altered nutritional supply of tryptophan during
development was also reported to cause changes in the brain
concentrations of tryptophan and serotonin in animal models
(Huether 1984
). There is commercial interest in
fortifying infant formulas with crystalline tryptophan, to simulate
more closely the amino acid composition of human milk
The objectives of this investigation were to determine the
bioavailability of tryptophan in cow's milk-based infant formulas
by using a rat growth method (Nielsen et al. 1985
) and
to study the effects of tryptophan supplementation on the levels of
brain tryptophan and serotonin in the rat model. The powdered and/or
liquid concentrate forms of infant formulas obtained from four
manufacturers were studied.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Samples of powdered and/or liquid concentrate forms of cow's milk-based infant formulas from four manufacturers were purchased locally during May of 1995. The protein sources used in the preparation of the formulas (as declared on the label) were as follows: manufacturer 1, powder: skim milk powder; manufacturer 1, liquid concentrate: whey powder (demineralized); manufacturer 2, powder: skim milk; manufacturer 2, liquid concentrate: evaporated skim milk; manufacturer 3, powder: reduced minerals whey and skim milk; manufacturer 3, liquid concentrate: skim milk and reduced minerals whey; manufacturer 4, liquid concentrate: skim milk and skim milk powder. All the liquid concentrates were lyophilized (12-L condensate capacity freeze dryer, Virtis, Gardiner, NY) before analyses and feeding to rats. Powders 1, 2 and 3 contained 11.81, 11.75 and 11.69% protein (dry basis), respectively. Similarly, liquid concentrates 1, 2, 3 and 4 contained 11.37, 11.94, 12.37 and 12.13% protein (dry basis), respectively.
Feeding studies.
A total of three rat feeding studies (expts. 1, 2 and 3) were conducted in this investigation. The first two experiments dealt with the determination of tryptophan bioavailability, whereas the third experiment dealt with the influence of tryptophan fortification of infant formulas on the levels of brain tryptophan and serotonin in rats.
In the first experiment, the dietary conditions for studying the
bioavailability of tryptophan by using a rat growth assay
(Nielson et al. 1985
) were standardized. This
included the feeding of a tryptophan-deficient basal diet and seven
tryptophan-supplemented diets. The basal diet was formulated to be
adequate in all nutrients for rat growth except tryptophan (Table 1
). The tryptophan-deficient diet was supplemented with seven graded
levels (0.02, 0.04, 0.06, 0.08, 0.10, 0.12 and 0.14%) of
L-tryptophan. A casein control diet (AIN
1977
) was also included in this experiment. All the
experimental diets were made isonitrogenous by the addition of a
mixture (1:1:1) of L-glutamic
acid:L-serine:glycine.
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In the third experiment, a protein-free basal diet was formulated
to contain (g/kg diet): soybean oil, 100; coconut oil, 100; cellulose,
50; minerals (AIN 1977
), 35; vitamins (AIN
1980
), 10; choline bitartarate, 2; lactose, 390; sucrose, 209
and cornstarch, 104. Two liquid concentrates (manufacturers 2 and 4)
and casein + L-methionine (0.2% of diet) were added to the
protein-free basal diet as the sole source of protein to provide
8% protein diets. The levels of soybean oil, coconut oil, lactose,
sucrose and cornstarch were varied to make all the diets equal in fat
(20%), lactose (39%) and energy (19.3 kJ/g). Each of the two test
protein diets (liquid concentrates 2 and 4) were supplemented with four
levels of L-tryptophan (0.0, 0.1, 0.5 and 1.0%). All the
nine protein-containing experimental diets were made isonitrogenous
by the addition of a mixture of nonessential amino acid
(L-glutamic acid:L-serine:glycine, 1:1:1). A
total of 10 experimental diets (nine protein diets plus one
protein-free diet) were tested in this experiment.
Male weanling (45 ± 5 g) CD Sprague-Dawley rats (Charles
River Canada, St. Constant, Quebec) were used in all the three
experiments. Each experiment was a completely randomized design. Rats
(n = 6 in expts. 1 and 2, and n
= 8 in expt. 3) were housed individually in a temperature- and
humidity-controlled facility (Sarwar 1997
). In all
the three experiments, rats were given free access to water and food
for 2 wk, and records of weekly food consumption and body weights were
kept. In expt. 3, after 2 wk of feeding, animals were killed while
anesthetized with 3% isoflurane in O2. On the day of the
kill, food was removed from the cages at 0700 h, and the rat kill
started at 1100 h. Blood samples were immediately collected from
the dorsal aorta of the rats into heparinized tubes, and plasma was
separated by centrifugation at 3000 x g for 15
min. Plasma samples were stored at -80°C until analyzed. The whole
brains were quickly removed, weighed and rapidly frozen in liquid
nitrogen. The frozen tissues were stored at -80°C until analyzed.
The collection of blood and brain samples was completed in 2 h.
This delay in collection was balanced across treatment groups. The
protocol was approved by the animal care committee of Health Canada,
and the rats were maintained according to guidelines of the Canadian
Council on Animal Care.
Analytical methods.
Total nitrogen concentrations of protein sources and diets were
determined by the microKjeldahl method by using a Kjeltec Auto 1030
Analyzer (Tecator, Herndon, VA). Protein was calculated by using a
nitrogen-to-protein conversion factor of 6.25. Protein sources were
hydrolyzed with 4.2 mol NaOH/L for the quantitative recovery of
tryptophan (AOAC 1990
). Tryptophan in the alkaline
hydrolysates was determined by using a simple liquid chromatography
method requiring no derivatization (Sarwar et al. 1988
).
Tryptophan concentrations in the plasma and brain samples, and
serotonin and 5-hydroxyindole-3-acetic acid
(5-HIAA)2
concentrations in the brain samples, were determined by the method of
Murai et al. (1988)
, by using liquid chromatography with
electrochemical detection.
Tryptophan bioavailability and protein quality determinations.
Food conversion efficiencies (FCE, g weight gain/g food consumed) were
calculated in expts. 1 and 2. Based on FCE, tryptophan bioavailability
was calculated by using a slope-ratio method (Finney 1971
). In expt. 3, protein efficiency ratio (PER), net protein
ratio (NPR) and relative NPR (RNPR) were calculated by using the
following equations (Sarwar 1997
):
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Statistical analysis.
In each experiment, data were analyzed by one-way ANOVA and
Tukey's honestly significant difference test (Steele and Torrie 1980
) by using the Statistical Systems for Personal Computers
(SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The diets tested in this experiment contained 28.73–29.15% protein.
Treatments had significant (P < 0.0001) effects on
weight gain, food consumed and FCE (Fig. 1
). Addition of the supplemental L-tryptophan to
the tryptophan-deficient basal diet resulted in improved weight
gain, food consumed and FCE in rat (Fig. 1)
. The positive response
peaked at 0.10% supplemental tryptophan and then declined at the
highest two levels of tryptophan (0.12 and 0.14%). The growth and FCE
means for the basal + 0.10% tryptophan group were not significantly
(P > 0.05) different from those of the rats fed the
casein control diet.
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were linear, giving validity to the results of the
second experiment. Experiment 2.
Treatments had significant effects on rat weight gain (P
< 0.0001) (Fig. 2
), food consumed (P < 0.001) (Fig. 3
) and FCE (P < 0.001) (Fig. 4
). Addition of the two levels (0.04 and 0.08%) of the
supplemental tryptophan (both from a crystalline source and from infant
formulas) resulted in improved rat growth and FCE (Figs. 2
and 4)
. As
expected, the data for the higher level (0.08%) of tryptophan
supplementation resulted in significantly (P < 0.05)
higher means those for rats fed the lower level of tryptophan (0.04%).
The models used in calculating slopes (based on a two-point
analysis, basal + 0.04% tryptophan and basal + 0.08% tryptophan) are
shown in Table 2
. The relative potencies (tryptophan bioavailability values) are also
shown. The tryptophan bioavailabilities (83–84%) for liquid
concentrates 2 and 4 were significantly (P < 0.05)
lower than those for other infant formulas. Among the powders, powder 2
had the lowest tryptophan bioavailability (90%).
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Treatments had significant effects on weight gain (P < 0.0001), food consumed (P < 0.01), PER (P
< 0.001), NPR (P < 0.001) and RNPR (P
< 0.001) (Table 3
). The protein quality, as predicted by PER, NPR and RNPR, of
liquid concentrates 2 and 4 was significantly (P < 0.05) inferior compared to that of the casein control diet (Table 3)
.
The protein quality of liquid concentrate 4 was significantly
(P < 0.05) lower than that of liquid concentrate 2.
The addition of tryptophan (0.1, 0.5 and 1.0%) had no effect on
protein quality of the two liquid concentrates (Table 3)
.
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). The concentrations of tryptophan in the plasma and brains
of rats fed the unsupplemented liquid concentrates were significantly
(P < 0.05) lower than of those of rats fed the casein
control diet (Table 4)
. Addition of tryptophan to the liquid
concentrates caused significant (P < 0.05) elevations
in the concentrations of plasma and brain tryptophan. The increases in
the levels of plasma and brain tryptophan corresponded to the amount of
supplemental tryptophan (0.1, 0.5 and 1.0%). A positive correlation
between plasma- and brain-tryptophan concentration was observed
(r2 = 0.98). The concentrations
of brain serotonin and 5-HIAA in rats fed the unsupplemented liquid
concentrates were also lower than in those fed the casein control diet
(Table 4)
. Tryptophan supplementation (0.1, 0.5 and 1.0%) of the
liquid concentrates resulted in significantly (P < 0.05) increased levels of brain serotonin and 5-HIAA. The increase was
most marked at the highest level of supplementation. There was a
positive correlation between brain 5-hydroxyindoles (sum of serotonin
and 5-HIAA) and brain tryptophan (r2 =
0.88) (Fig. 5
) or plasma tryptophan (r2 =
0.97).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which would
confirm that the basal diet was adequate in all nutrients except
tryptophan for rat growth. The FCE of rats fed diets containing
0.00–0.08% supplemental L-tryptophan (Fig. 1)
was linear,
further validating to the methodology used to determine tryptophan
bioavailability in expt. 2 (Table 2)
.
Powders 1, 2, and 3 and liquid concentrates 1, 2, 3 and 4
contained 13.5, 12.3, 13.8, 12.0, 11.8, 13.3 and 11.6 g
tryptophan/g protein, respectively. Based on the tryptophan
bioavailability data (Table 2)
, the levels of bioavailable tryptophan
in the three powders and the four liquid concentrates would be 12.7,
11.1, 13.1, 11.0, 9.8, 12.6 and 9.7 mg/g protein, respectively. The
levels of bioavailable tryptophan in these formulas (9.7–13.1 mg/g
protein) are considerably lower than the level of tryptophan (17–19
mg/g protein) in human milk (FAO-WHO 1991
, Sarwar et al. 1996
). The generally lower levels of bioavailable
tryptophan in liquid concentrates compared to powders may be due to the
use of more severe heat treatment in the preparation of liquid
concentrates compared to powders (Packard 1982
). Liquid
concentrates were reported to have considerably lower protein
digestibilities and qualities than powders prepared by the same
manufacturer (Sarwar et al. 1989
).
The third experiment was designed to estimate protein quality by
using the RNPR method (2 wk), which is the most appropriate rat test
for routine evaluation of protein quality (Codex Alimentarius Commission 1984
). For comparison, the 2-wk PER values were
calculated (Table 4)
. Significant correlations
(r2 = 0.90, P < 0.01)
were reported between 2-wk PER or NPR and 4-wk PER values of milk- and
soy-based infant formulas (Harris and Burns 1988
).
Because casein is limiting, for rat growth, in sulfur amino acids
(AIN 1977
, Reeves et al. 1993
), the RNPR
method includes the use of the methionine-supplemented Animal
Nutrition Research Council casein as the reference protein. PER
and NPR are commonly determined at the 10% protein level. However,
several researchers have used 8 or 9% dietary protein in determining
PER or NPR values of protein products (Henry 1965
,
Lowry and Baker 1989
, Sammonds and Hegsted 1977
). Diets formulated to contain 8% protein were used in the
present study because the proteins used as control, such as egg
and methionine-supplemented casein, support peak PER and NPR values
at about that level of dietary protein (Henry 1965
,
Sarwar and McLaughlan 1981
).
The protein quality data obtained in this investigation (Table 3)
cannot be applied directly to infants because of the differences in
amino acid requirements of rats and infants. Therefore, the protein
quality data obtained with rats (Table 3)
should be seen as an
indication for the direction for further research. The inferior
protein quality of liquid concentrate 4 compared to liquid concentrate
2 (Table 3)
may be due to its lower level of digestible methionine +
cystine (21 vs. 28 mg/g, concentrate 4 vs. 2, respectively), the first
limiting amino acid in infant formulas for rat growth (Sarwar et al. 1989
).
Tryptophan supplementation (0.1, 0.5 and 1.0%) of liquid concentrates
2 or 4 had no adverse or beneficial effects on rat growth or protein
quality indices (Table 3)
. Similar observations have been made about
the safety of L-tryptophan in pigs fed diets supplemented
with 0.1 and 1.0% L-tryptophan (Chung et al. 1991
). Higher levels of L-tryptophan
supplementation (2 and 4% of diets) were, however, reported to
decrease weight gains and food intake in pigs (Chung et al. 1991
). Feeding low-protein diets (6 or 10% casein)
containing 5% supplemental L-tryptophan resulted in
reduced food intake and markedly decreased weight gains in rats
(Muramatsu et al. 1971
). The growth depressing effect of
5% supplemental L-tryptophan was counteracted completely
by increasing the dietary protein concentration to 25%
(Muramatsu et al. 1971
).
Tryptophan supplementation (0.1, 0.5 and 1.0%) of liquid concentrates
2 or 4 resulted in a dose-related increase in the levels of plasma
tryptophan and of brain tryptophan and 5-hydroxyindoles (serotonin and
5-HIAA) in rats (Table 4)
. Similarly, dose-related increases in the
levels of brain tryptophan and 5-hydroxyindoles of rats were reported
when a low-protein diet (5% whole egg protein) was supplemented
with 0.22 and 0.52% L-tryptophan (Yokogoshi et al. 1987
). Serotonin synthesis in the brain of rats
(Fernstrom and Wurtman 1971
), monkeys (Leathwood and Fernstrom 1990
) and dogs (Moir and Eccleston 1968
) was also reported to be influenced by the availability of
L-tryptophan.
The level of brain tryptophan is dependent not only on plasma
tryptophan but also on the ratio of plasma tryptophan to the sum of
other large neutral amino acids (LNAA) such as phenylalanine, tyrosine,
leucine, isoleucine and valine, which compete for a common brain
transport system (Fernstrom and Wurtman 1972
). The
plasma levels of the LNAA, other than tryptophan, were not determined
in the present investigation. The diets (expt. 3) were made
isonitrogenous by the addition of a mixture of L-glutamic
acid, L-serine and glycine. Because the addition of these
dispensable amino acids would not influence plasma levels of LNAA, we
assumed that the dietary tryptophan-induced increases in plasma
tryptophan (Table 4)
would have also resulted in increased plasma
tryptophan/LNAA ratios.
Even the lowest level of supplemental tryptophan (0.1% of diet or
~12 mg/g dietary protein) caused significant increases in the levels
of plasma and brain tryptophan and brain serotonin and 5-HIAA (Table 4)
. By using tryptophan bioavailability values of 83 and 84% for
liquid concentrates 2 and 4, respectively (Table 2)
, we calculated that
the two liquid concentrates, at this level of supplementation, would
contain ~20 mg/g protein of bioavailable tryptophan. This level of
bioavailable tryptophan may be comparable to the level in human milk
(17–19 mg/g protein) (FAO-WHO 1991
, Sarwar et al. 1996
) because supplemental crystalline tryptophan may be
more susceptible to heat treatment than is the protein-bound
tryptophan in the milk-based infant formulas
(Fazzolari-Nesci et al. 1992
). Therefore, we suggest
that supplementing the liquid concentrate forms of milk-based
infant formulas with L-tryptophan to raise the level of
tryptophan to that of the human milk would increase the levels of brain
serotonin and 5-HIAA in infants fed the tryptophan fortified
formula. This may have beneficial effects on infant sleep
latency, which could influence neurobehavioral development
(Steinberg et al. 1992
). The present study supports the
need to investigate the effects of tryptophan supplementation of infant
formulas, to simulate the tryptophan composition of human milk, on
sleep latency and neurobehavioral development in infants.
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FOOTNOTES |
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Manuscript received November 6, 1998. Initial review completed January 22, 1999. Revision accepted May 19, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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