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* Department of Neonatology University of Genova, G. Gaslini Institute,
Obstetrics and Gynecology, Universiteit Maastricht, Maastricht, The Netherlands and
** General Pathology and
## Department of Pediatrics, G. Gaslini Institute, Genova, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • artificial feeding • nucleotides • erythrocyte • oxygen affinity • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This results in a diminished
affinity of hemoglobin for oxygen, leading to enhanced peripheral
oxygen release at a given oxygen tension. In conditions associated with
decreased oxygen availability, such as high altitude, chronic lung
disease or cyanotic heart disease, the concentration of erythrocyte
2,3-DPG is physiologically augmented to compensate for the decrease in
peripheral oxygen delivery (Oski et al. 1969
). The rise
in erythrocyte 2,3-DPG concentration, associated with the increase in
peripheral oxygen supply, might offer new therapeutic perspectives in
conditions that are associated with a compromised peripheral metabolic
environment. An example is intrauterine growth-retarded neonates who
have been described as having a compromised metabolic environment and
decreased erythrocyte 2,3-DPG concentrations (Farquharson 1983
, Siegel et al. 1979
). Previously, it was
demonstrated that the concentration of 2,3-DPG can be increased in
isolated erythrocyte when nucleosides (adenosine and/or inosine) are
added to the incubation medium (Akerblom et al. 1968
,
Dawson et al. 1971, Hogman et al. 1973
).
This can be ascribed to a biochemical pathway involving a
phosphorolytic cleavage to hypoxantine and ribose 1-phosphate which led
to a rise in 2,3-DPG production through pentose-shunt reactions and
anaerobic glycolisis.
Whether dietary intake of nucleotides also increases erythrocyte
2,3-DPG is unknown. It has already been demonstrated that dietary
nucleotides are degraded to nucleosides by pancreatic nucleases,
intestinal phosphoesterases and especially by the intestinal alkaline
phosphatase (Munro 1984
). Moreover, administration of
nucleotide-supplemented-formula-milk to neonates leads to increased red
cell phospholipid content (De Lucchi et al. 1987)
enhanced immunity (Carver et al. 1991)
and to better
intestinal growth and maturation (Uauy et al. 1990)
.
This raises the question if administration of nucleotide-supplemented
milk to neonates leads to an increase in their erythrocyte 2,3-DPG
concentration. To this end, we hypothesize that the erythrocyte 2,3-DPG
concentration can be increased by feeding neonates
nucleotide-supplemented formula or milk. The study was performed in
three groups of rat pups that were either breast-fed (C) or
artificially fed with formula which was either supplemented with
nucleotides (S) or was nucleotide-free (NS).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.Sprague-Dawley rat pups (n = 42), and their dams
(n = 4), were supplied by a commercial breeder
(Charles-River Laboratories, Zurich, Switzerland) 5 d after birth.
All facilities and procedures were approved by the Institutional Animal
Care and Use Committee of the University of Genova. Pups taken from the
same litter were numbered and progressively assigned to three groups,
group C (n = 14), NS (n = 14) and S
(n = 14). The back of each animal was marked with
colored dye to distinguish the various groups and to allow putting the
pups with their own mothers during the night throughout the
experimental period. Afterward the animals were allowed acclimation to
the centralized experimental animal facilities until d 9 postpartum
with a 12 h/12 h light/dark cycle. The dams were given free access to
standard nonpurified diet.
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On d 9, seven pups from each group were randomly weighed and killed by
decapitation. Blood was collected from the heart (1 mL) to measure
2,3-DPG, hematocrit (Hct) (microcapillary method) and hemoglobin (Hb)
(OSM; Radiometer, Copenhagen, Denmark) concentrations. In some pups the
amount of blood that was obtained from the heart after decapitation was
too small to perform analysis on Hct and Hb. Until d 16 after birth,
group C was breast-fed. Groups NS and S were separated from their
mothers every day at 0900 h until d 16. They were fed every 3 h by slow intragastric injection with formulas, which was either S or
NS. The amount of injected fluid was calculated following neonatal
artificial rat feeding curves Messer et al. (1969)
and
was gradually increased during the experimental period from 0.75 to 1.5
mL per injection. This depended on the neonatal weight gain and the age
of the pups and was decided to avoid gastric overdistension. Studies
based on solely artificial intake were previously paralleled by an
increased risk of death by overdistension and increased intestinal gas
formation (Dymsza et al. 1964
, Miller and Dymsza 1963
). Therefore, to obtain a high survival rate, the pups were
placed with their dams at 2000 h to allow suckling. On d 16 all
pups were weighed and killed by decapitation. Blood was collected from
the heart (~1 mL) to measure 2,3-DPG, Hct and Hb concentrations. The
2,3-DPG/Hb ratio, reflecting the affinity of Hb for oxygen, was
calculated for each rat pup.
Assays.
Erythrocyte 2,3-DPG concentration was measured by quantitative
enzymatic determination with Diagnostics (St. Louis, MO) 2,3-DPG acid
reagents. Freshly obtained heparinized blood (1 mL) was mixed with cold
trichloroacetic acid (3 mL) and was shaken vigorously for 3 s then
centrifuged (10 min, 3000 x g). The supernatant
(2,3-DPG in tricholoroacetic acid) was enzymatically hydrolyzed to
3-phosphoglycerate and inorganic phosphorus by the 2,3-DPG phosphatase
activity associated with the enzyme phosphoglycerate mutase. As
previously described (Keitt 1971
), liberated phosphorus,
the oxidation of NADH to NAD reflects the concentration of DPG
originally present. Determination of 2,3-DPG as described above had a
2.2% CV.
Statistics.
The results are presented as median, 75th and 25th percentile throughout the text unless otherwise stated. Differences between d 9 and d 16 in each group were evaluated by Wilcoxon-Ranked-Sum-Test. Differences among groups C, NS and S were evaluated using the Mann-Whitney-U-test. A P value of <0.05 (two-sided) was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.Erythrocyte 2,3-DPG concentration and neonatal weight increased in all
groups by d 16 compared to d 9. The 2,3-DPG concentration at d 9 did
not differ among the groups while at d 16 it was significantly lower in
the NS group than in the C- and S groups (P < 0.05).
In C and NS groups both Hct and Hb were greater at d 16 than at d 9
(P < 0.05). The difference was not significant in the
S group, though this may be related to the relatively limited number of
observations. The change in erythrocyte 2,3-DPG concentration was
significantly greater in the S group than in the NS group
(P < 0.05) (Fig. 2
).In the NS rat pups the 2,3-DPG/Hb ratio was significantly lower at d 16
than at 9 and was lower than in the C and S pups (Fig. 3
;P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Baumann et al. 1973
, Blunt et al. 1971
, Noble et al. 1983
), the 15% rise in 2,3-DPG observed in the C and NS groups
was considered to be a normal physiological phenomenon. Taking this
physiological 2,3-DPG postnatal increase into account, the additional
10% increase in erythrocyte 2,3-DPG observed in the S group likely can
be attributed to nucleotide supplementation. Therefore, since no data
concerning the nucleotide profile of rat milk are available, we can
hypothesize that it is low in nucleotides.
The rises in Hct and Hb were also considered physiological since the
pattern of changes between d 9 and 16 were comparable among the three
groups and were observed by others in different species
(Menendez-Patterson et al. 1987
, Mortola et al. 1986
, Styka and Penney 1977
). Supplementation of
nucleotides to the neonatal rat pups did not cause additional effects
on the postnatal rise of Hct and Hb.
In the first month of life, human neonate Hb levels and Hct decrease,
while DPG erythrocyte concentration remains stable or slightly
increases (Delivoria-Papadoupolos et al. 1971
). As in
adults (Hielm 1969
, Torrance et al. 1970
), this "physiological neonatal anemia" is probably
compensated for by an increased DPG/Hb ratio that maintains an adequate
oxygen delivery to the tissues by increasing the pO2 at
which the blood reaches the 50% saturation (P50).
Therefore, Hb concentration is negatively correlated with
P50 values (Koizumi 1991
, Samaja et al. 1990
).
Unfortunately, the aggressive sampling procedure and the small amount of blood that was obtained from each pup did not allow us to measure other variables (pH, pCO2, HbF%), that may affect the position of the oxygen dissociation curve.
However, in these rat pups, despite a physiological Hb and Hct increase, the additional increase in 2,3-DPG obtained in the S group prevented the drop in the 2,3-DPG/Hb ratio, which was observed in the NS group. This suggests that in the S group, artificial feeding with nucleotide-supplemented formula is associated with a higher peripheral oxygen supply.
Not surprisingly, in this particular experiment, the increases in
the erythrocyte 2,3-DPG concentration and the 2,3-DPG/Hb ratio were not
paralleled by a greater increase in neonatal weight in the S group.
Theoretically, a rise in erythrocyte 2,3-DPG would have a more profound
effect on neonatal weight gain and on the metabolic performance of
neonates having a compromised peripheral metabolic environment and
decreased erythrocyte 2,3-DPG values as a result of either an unpaired
intrauterine growth and/or altered lung function subsequent to neonatal
respiratory distress syndrome (Farquharson 1983
,
Siegel et al. 1979
). This is confirmed by other results
(Cosgrove et al. 1996
) describing improved catch-up
growth after nucleotide administration, in small for gestational age
infants whose intestinal mucosa was hypothesized to be functionally
impaired as a result of undernutrition.
Currently the effect of an increase in 2,3-DPG, particularly on the
peripheral hemodynamic and metabolic environment in neonates, cannot be
deduced from either the present study or from other studies
(Cosgrove et al. 1996
). However our experimental data
may provide essential background for performing clinical investigations
in human newborns.
From the present study we can conclude that erythrocyte 2,3-DPG concentration can be increased by dietary supplementation of nucleotides in neonatal rats. As hypothesized above, it remains to be seen whether artificial formulas enriched with nucleotides might provide positive effects on the neonatal pathological conditions (respiratory distress syndrome, bronchopulmonarydysplasia) in which more oxygen release to peripheral tissues is required. Further experimental and clinical nutritional investigations are needed to achieve this goal.
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FOOTNOTES |
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1 Supported in part by a SIP (Italian Society of
Pediatrics) fellowship.
2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Abbreviations used: C, breast-fed rat pups;
2,3-DPG, 2,3-diphosphoglycerate; HbF, fetal hemoglobin; Hct,
hematocrit; NS, rat pups fed a nucleotide-free formula;
P50, O2 pressure at which 50% of blood is
saturated; S, rat pups fed a nucleotide-supplemented formula.
Manuscript received October 21, 1998. Initial review completed November 3, 1998. Revision accepted November 25, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Akerblom O., De Verdier C.X.H., Garby L., Hogman C. Restoration of defective oxygen transport function of stored red blood cells by addition of inosine. Scand. J. Clin. Lab. Invest. 1968;21:245-247[Medline]
2. Bartels H., Bartels R., Ratschlag-Schaefer A. M., Robbel H., Ludders S. Acclimatization of newborn rats and guinea pigs to 3000 to 5000 M simulated altitudes. Respir. Physiol. 1979;36:375-389[Medline]
3. Baumann R., Teischel F., Zoch R., Barterls H. Changes in red cell 2,3-diphosphoglycerate concentration as cause of the postnatal decrease of pig blood oxygen affinity. Respir. Physiol. 1973;19:153-161[Medline]
4. Benesh R., Benesh R. Intracellular organic phosphates as regulators of oxygen release by haemoglobin. Nature 1969;221:40-46[Medline]
5. Blunt M. H., Kitchens J. L., Mayson S. M., Huisman T.H.J. Red cell 2,3-diphosphoglycerate and oxygen affinity in newborn goats and sheep (35993). Proc. Soc. Exp. Biol. Med. 1971;138:800-803[Medline]
6.
Carver J., Pimentel B., Cox W. I., Barness L. A. Dietary nucleotides upon immune function. Pediatrics 1991;88:359-363
7. Cosgrove M., Davies D. P., Jenkins H. R. Nucleotide supplementation and the growth of term small for gestational age infants. Arch. Dis. Child. 1996;74:F122-F125
8. Delivoria Papadopoulos M., Miller W. W. Red cell 2,3 diphosphoglycerate levels in subject with chronic hypoxemia. N. Engl. J. Med. 1969;280:1165-1166
9. Delivoria-Papadopoulos M., Roncevic N. P., Oski F. A. Postnatal changes in oxygen transport of term premature, and sick infants: The role of red cell 2,3-diphosphoglycerate and adult hemoglobin. Pediatr. Res. 1971;5:235-245
10. De Lucchi C., Pita M. L., Faus M. J., Molina J. A., Uay R., Gil A. Effects of dietary nucleotides on the fatty acid composition of erythrocyte membrane lipids in term infants. J. Ped. Gastroenterol. Nutr. 1987;6(4):568-574[Medline]
11. Dymsza H. A., Czajka D. M., Miller S. A. Influence of artificial diet on weight gain and body composition of the neonatal rat. J. Nutr. 1964;84:100
12. Farquharson R. G. Fetal oxygen affinity and its parameters in a random obstetric population. J. Perinat. Med. 1983;11:43-50[Medline]
13. Hielm M. The content of 2,3 diphosphoglycerate and some other phosphocompounds in human erythrocytes from healthy adults and subjects with different types of anaemia. Forsvarsmed 1969;5:219-226
14. Hogman, C. F., Akerbloom, O. & Artuson, G.(1973)Experience with new preservatives: summary of experiences in Sweden. In: The Human Red Cell In Vitro (Grune & Stratton, eds.), pp. 217. New York.
15. Keitt A. S. Reduced nicotinamide adenine dinucleotide-linked analysis of 2,3-diphosphoglyceric acid: Spectrophotometric and fluorometric procedures. J. Lab. Clin. Med. 1971;77:470-475[Medline]
16. Koizumi M. Oxyhemoglobin dissociation curve and 2,3 dyphosphoglycerate in chronic hypoxemia. Nippon Kyobu Shikkan Gakkai Zasshi 1991;29(5):547-553
17. Menendez-Patterson A., Fernandez S., Diaz F., Marin B. Malnutrition in rats during pregnancy and lactation period: a study on body, spleen and thymus weights and hematologic parameters in dams and their offspring. Revista Espanola de Fisiologia 1987;43(3):287-296[Medline]
18. Messer M., Thoman E. B., Galofre Terrasa A., Dallman P. R. Artificial feeding of infant rats by continuous gastric infusion. J. Nutr. 1969;98:404-410
19.
Miller S. A., Dymsza H. A. Artificial feeding of neonatal rats. Science 1963;141:517-518
20.
Mortola J. P., Morgan C. A., Virgona V. Respiratory adaptation to chronic hypoxia in newborn rats. J. Appl. Physiol. 1986;61(4):1329-1336
21. Munro H. N. Differences in metabolic handling of orally versus parenterally administered nutrients. Greene M. Greene H. L. eds. the Role of the Gastrointestinal Tract in Nutrient Delivery 1984:183-186 Academic Press New York.
22.
Noble N. A., Jansen C.A.M., Nathanielsz P. W., Tanaka K. R. Mechanism of red cell 2,3-disphosphoglycerate increase in neonatal lambs. Blood 1983;61(5):920-924
23. Oski F. A., Gottlieb A. J., Dawson R. B., Edinger M. C., Ellis T. J. Hemoglobin function in stored blood. J. Lab. Clin. Med. 1971;77:46-53[Medline]
24. Samaja M., Rovida E., Motterlini R., Tarantola M., Rubinacci A., de Prampero P. E. Human red cell age, oxygen affinity and oxygen transport. Respir. Physiol. 1990;79(1):69-79[Medline]
25. Siegel S. R., Oh W., Fisher D. A. Fructose-1,6-diphosphatase and glucose-6-phosphatase in newborn rats with intrauterine growth retardation. Early Hum. Dev. 1979;3(1):43-49[Medline]
26. Styka P. E., Penney D. G. The perinatal rat: body weight, hematocrit and regional changes in heart weight and lactate dehydrogenase isoenzyme composition and activity. Growth 1977;41:325-336[Medline]
27. Torrance J., Jacobs P., Restrepo A., Eschbach J., Lenfant C., Finch C. A. Intraerythrocytic adaptation to anemia. N. Engl. J. Med. 1970;283:165-169
28. Uauy R., Stringel G., Thomas R., Quan R. Effect of dietary nucleosides on growth and maturation of developing gut in the rat. J. Pediatr. Gastroenterol. Nutr. 1990;10(4):497-503[Medline]
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