SYMPOSIUM
PROCEEDINGS
Nurn’tion Vol. 13. No. 5. 1997
Dietary Nucleotides May Influence
the Humoral Immunity in
Immunocompromised Children
OLGA MARTiNEZ-AUGUSTIN,
ANTONIO MARTINEZ-VALVERDE,
From the Departamento
PHD, JULIO J. BOZA, PHD, JOAiQUIN NAVARRO, PHD,
MD, MAGDALENA ARAYA, MD, AND ANGEL GIL, PHD
de Bioquimica y Biologia Molecular,
Dietary nucleotides ( NTs) are considered nonessential nutrients, but under certain conditions may behave as semi-essential.
This means that they may become essential when the exogenous
supply is insufficient for normal function, even though their
absence from the diet does not lead to a classic clinical deficiency syndrome.’
Recent studies have examined the effects of dietary NTs on
the gut, showing that they may be important in achieving optimal gut development
and growth, especially after intestinal
injury.‘mh In addition, there is evidence of the potential role of
exogenous NTs as modulators of the immune function.7m’4
In recent years we have carried out a series of studies in
order to determine the influence of dietary NT supplementation
on the intestinal absorption of macromolecules,
intestinal permeability, serum immunoglobulin
(Ig) levels, and the development of circulating antibodies against /?-lactoglobulin
and (Ycasein in preterm infants for the first month of life. Moreover,
additional studies have been carried out to determine their influence on the recovery of malnourished
and infected children
with diarrhea.
Twenty-seven
preterm infants born between 32 and 36 wk
of gestational age (which was estimated according to the mother’s last period), weighing between 1500 and 2200 g were
enrolled in the present study upon admission to the Hospital
Matemo Infantil of Malaga, Spain. All infants were free from
major neonatal disease and were able to tolerate enteral feeding
within 48 h after birth. Infants were assessed at 1, 7, and 30 d
of age, when blood and urine samples were obtained after the
administration of a dose of lactulose and mannitol (300 and 60
Universidad de Granada, Granada, Spain
mg, respectively)
dissolved in the liquid diet. Eleven infants
were fed a standard low-birth-weight
infant formula (F) and
16 infants were fed the same formula supplemented
with NTs
(FN) for 30 d.*
Lactulose and mannitol in urine were determined by gasliquid chromatography
following the method previously deet ~1.‘~ and P-lactoglobulin
sescribed by Martinez-Augustin
rum concentration
was measured by a modification
of the
ELISA method described by Husby et ul. I6
There were no significant differences between the lactulose/
mannitol ratios in the urine of children fed F or FN at any of
the timepoints considered in the study. While the lactulose/
mannitol ratio was the same or slightly higher in infants fed FN
during the first week of life, this ratio decreased significantly at
30 d of life in both groups (Fig. 1).
Intact P-lactoglobulin
was detected in the serum of all infants included in the study at 1,7, and 30 d of life. Nevertheless,
there were no significant differences either timewise or between
the formulas used (Fig. 1) .
Thus, in this study we could confirm the presence of enhanced intestinal permeability in preterm infants during the first
week of life, whereas gut closure was effective at 30 d of life.
Although it has been shown that dietary NTs have relevant
effects on the intestinal growth and rate of maturation in young
rats as assessed by mucosal weight, RNA and DNA content,
protein concentrations,
and activity of brush border enzymes,4
our data showed that NT supplementation
of the low-birthweight infant formula did not lead to any change in the intestinal
permeability in the preterm infant.
* Feeding regimens: All infants were fed according to their appetite and tolerance to assure an intake of at least 100 kcal. kg-’ .d-’ by 10 d of
life. Feedings were increased for infants who appeared hungry between meals and temporarily reduced or discontinued
in the presence of gastric
residuals greater than 2-3 mL, vomiting, or abdominal distention. Milk formulas contained 2 g/l00 kcal protein (60% whey protein, 40% casein),
5.4 g/ 100 kcal fat (17.7% medium-chain
triglycerides,
25.0% milk fat, 43.8% olive oil, and 13.5% soy oil), 9.9 g/l00 kcal carbohydrates
(4.5 g/
100 kcal of lactose and 5.4 g/100 kcal dextrin-maltose),
and 0.6 g/ 100 kcal minerals and vitamins, in agreement with the ESPGAN (European
Society for Paediatric Gastroenterology
and Nutrition) recommendations.‘3
Both powder formulas provided 80 kcal/dL when diluted to 15% (wt/
vol). To obtain the FN formula,
100 g F diet was supplemented
with 1.12 mg citidine 5’-monophosphate
(CMP),
1.32 mg adenosine
5 ‘-monophosphate
(AMP), 1.49 mg guanosine 5 ‘-monophosphate
(GMP), 3.42 mg uridine 5 ‘-monophosphate
(UMP), and 0.45 mg inosine 5 ‘monophosphate
(IMP). When diluted to 80 kcal/dL, the FN formula contained
1.68 mg/L citidine 5’-derivatives,
1.98 mg/L adenosine 5 ‘derivatives, 2.23 mg/L guanosine 5 ‘-derivatives, 5. I3 mg/L uridine 5 ‘-derivatives, and 0.68 mg/L inosine 5 ‘-derivatives.
Nutrition 13:465-469.
1997
OElsevier Science Inc. 1997
Printed in the USA. All rights reserved.
ELSEVIER
0899.9007/97/$17.00
PI1 SO899-9007(97)00012-9
DIETARY NUCLEOTIDES’
a)
INFLUENCE
ON IMMUNITY
IN CHILDREN
1.2
1
0.8
g
@ 0.6
5
0.4
0.2
0
b)
1
7
30
1
ege (days)
Postnatal
7
30
Postnatalqe
(days)
B
6
PostnchI
age(days)
Postnatal
200
age
(days)
200
/*-
.
/
150
f
‘l-4
150
2E
Q
0
"'1
&
\
1
7
30
"
1
7
30
Postnatal age (days)
Postncld
age
(days)
Postnalti
age
(dqs)
FIG. 1. Urinary lactulose/mannitol (LM) ratio (a), concentration of serum P-lactoglobulin (b), and concentrations of serum IgG against a-casein
and P-lactoglobulin (c and d, respectively) in preterm infants at 1, 7, and 30 d of life fed a low-birth-weight standard milk formula (F) (n = 11)
(left column) and the same formula supplemented with nucleotides (FN) (n = 16) (right column). Results are expressed as mean t SEM. *P <
0.05; **P < 0.01 versus 1 d of postnatal age. “P < 0.05; lnP < 0.01 versus 7 d of postnatal age. Sop < 0.01 versus F.
DIETARY
NUCLEOTIDES’
INFLUENCE
ON IMMUNITY
IN CHILDREN
467
&A
mg/dl
q
ON
*
I
IF
1
20-30 days
3 months
mg/dl
mg/dl
W
so0
1207
C
a00
;li
700
I
/ CIFN
IF
/
so0
600
J3-.llI
400
300
200
100
0
-L
cord
bkmd
1octays
0
m
rJ=#
rmL8
T-
cud
blood
low
FIG. 2. IgA, IgG, and IgM concentrations
in pretetm infants’ cord blood and serum at 1, and 20-30 d, and at 3 mo of postnatal life, fed a lowbirth-weight
standard milk formula (F) (n = 12) and the same formula supplemented
with nucleotides (EN) (n = 12). Results are expressed as
mean ? SEM. *P < 0.05; **P < 0.01 versus F.
FN
F
FN
FIG. 3. IgG against a-casein and P-lactoglobulin
(3a and 3b, respectively)
levels in the serum of malnourished
and infected children before (A)
and after (B) being fed a standard milk formula (F) (n = 11) or the same formula supplemented
with nucleotides (FN) (n = 11). No statistical
differences were found. Results are expressed as mean ? SEM.
DIETARY
468
w
mg/ml
am
NUCLEOTIDES’
INFLUENCE
mglml
IgM
r-l
OA
ON IMMUNITY
3m-
IB
la33
IN CHILDREN
0
CIA
IB
ml
la
lm
lcoo
aal
ea,
4m
zll
0
FN
saliva IgA
n
OA
FN
mg/ml
I@
aT
m
FN
FIG. 4. Saliva IgA and serum IgG, IgA, and IgM in malnourished children before (A) and after (B) being fed a standard milk formula (F)
(n = 11) or the same formula supplemented with nucleotides (FN) (n = 11). Results are expressed as mean + SEM. *P < 0.05; **P < 0.01
versus A.
In the study described above, we tried to verify whether
NTs could affect the development of specific Igs against food
proteins. Thus we measured serum levels of IgG against
P-lactoglobulin
following the ELISA method described by
Hampton ef al., ” as well as serum levels of IgG against CXcasein following a similar protocol, except that the wells were
coated with cy-casein (10 pg/mL in carbonate/bicarbonate
buffer).
The concentration
of serum IgG against a-casein and ,&
lactoglobulin, the two main antigenic proteins present in infant
formulas based on cow’s milk, seemed to be stable during the
first week of life in our study. These values increased with
postnatal age, except in the case of the serum IgG against ,0lactoglobulin of those preterm infants fed the standard diet.
Preterm infants fed the NT-supplemented
diet generally
showed higher concentrations
of serum IgG against either (Ycasein or P-lactoglobulin
throughout the study, although these
differences were only significant in the case of the serum IgG
to P-lactoglobulin
at 30 d of life.
We also carried out another study in collaboration with the
Pediatric Policlinic of the University
Hospital of Granada,
aimed to evaluate the influence of dietary NT supplementation
on total serum Ig levels. This study included 24 healthy pretetm
infants, and a similar protocol to that described above was
followed, with 12 infants receiving F and 12 PN.
Serum samples were obtained at 10 and 20-30 d, and at 3
mo of life. Cord blood was also obtained. Igs in sera were
measured by nephelometry
using commercial kits purchased
from Behring (Marburg, Germany).
Cord-blood levels of IgG were lower than those described
DIETARY
NUCLEOTIDES’
INFLUENCE
ON IMMUNITY
for normal full-term infants, corresponding
to the low transfer
time of maternal IgG across placenta. During the first 3 mo of
life, a significant decrease in IgG was observed in both groups.
No differences in serum IgG levels were detected at any time
between both formula groups (Fig. 2).
IgM serum concentrations
showed a completely different
pattern. Low levels of this Ig were detected in the cord blood,
whereas a progressive increase of IgM concentration
with age
was observed. At 20-30 d as well as at 3 mo of life, higher
IgM concentrations
were detected in the FN group compared
with the F group.
We reported IgA in serum of infants only after 20-30 d of
life, because of the low content at early stages. Even at this time,
only 50% of infants in group F and 66% in group FN reached
measurable concentrations. All infants had higher levels of IgA
when they were 3 mo old, and at this age infants fed the formula
supplemented with NTs showed higher levels (P < 0.05) of this
Ig than those fed the nonsupplemented
formula.
The goal of the third study was to evaluate the influence of
dietary NT supplementation
on the recovery of infected and
malnourished children with diarrhea. Twenty children under 3
y of age (mean = 17.1 mo) admitted to the Hospital Albina R
de Patino (Cochabamba,
Bolivia) because of persistent diarrhea
( > 15 d) were included in the study. At the time of admission,
mean duration of diarrhea was 46.2 d and 37.3 d in the experimental and control groups, respectively,
and all the children
were malnourished
and infected. Etiologic studies in search of
IN CHILDREN
469
pathogens (bacteria, parasites, and rotavirus) demonstrated
a
total of 49 detections in the 22 patients. Children were randomly
assigned to a control group (F) (II = 11)) which received a
formula without NTs (NIEDAR, Puleva S.A., Granada, Spain),
or to an experimental
group (FN) (n = 1 1 ), which received
the same formula supplemented with NTs. Blood samples were
obtained at the beginning of the study and after 3 wk of recovery. Total IgG, IgA, and IgM and specific IgG against p-lactoglobulin and a-casein levels were measured using techniques
described above. Refeeding after malnutrition did not produce
any significant changes in either specific or total serum IgG
concentrations
or in saliva IgA levels (Figs. 3 and 4). Serum
IgA decreased significantly during the refeeding period in the
group fed the NT-supplemented
formula (Fig. 4). IgM concentration followed an opposite pattern, and significant differences
were found in the group fed the formula without NTs (Fig. 4).
In summary, although NTs did not affect intestinal closure
in preterm infants, enhanced IgA and IgM levels of Igs were
detected in preterm infants fed a NT-supplemented
formula. In
addition, levels of IgG against P-lactoglobulin
were increased
in preterm infants fed the same NT-supplemented
formula.
From our results we can conclude that dietary NTs may affect
Ig production
in immunocompromised
children, but further
studies are needed to determine the possible mechanisms
of
action of this effect and to clearly define the patterns of alterations that dietary NTs produce on the immune response in
vivo.
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