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1 Preterm Delivery

Preterm delivery in humans, defined as birth before 90 % gestation, is a leading cause of infant morbidity and mortality worldwide, occurring in approximately 10 % of all pregnancies (McIntire et al. 1999). Infants born prematurely account for the majority of all neonatal deaths. Not surprisingly, preterm infants show various signs of organ immaturity and this may make preterm neonates more sensitive to serious feeding-induced gastrointestinal complications (Siggers et al. 2011). The immature gastrointestinal tract is less able to deal with microbiology, immunology, and nutrition-related challenges of postnatal life as a result of deficiencies in intestinal structural integrity, digestive capacity, and intestinal immunity (Neu 2007). Such deficiencies are associated with increased enteric disease susceptibility in preterm versus term neonates. Thus, investigating means of improving these deficiencies will aid in improving the maturation of the preterm gastrointestinal tract, in reducing gut inflammation, and in optimizing nutrition and health in this compromised population.

2 Animal Models

However, less detailed information is available from human infants partly due to the difficulties in performing well-controlled studies on this vulnerable population of infants. In addition, it is neither ethical nor practical to conduct these experiments with the human fetus or infant.

Often, the more suitable approach is the use of animal models. While some earlier studies have focused on information derived mainly from rodent models (e.g., rats and mice) (Sodhi et al. 2008), it is difficult to conduct experiments with laboratory rodents because of their small body size and immature organs at birth, and this makes the large farm animals (e.g., pigs, cattle, sheep) more attractive models in this field.

Although no animal model will ever perfectly mimic the human condition, the pig has emerged as a superior non-primate experimental animal model because of similarities in anatomy, development, nutrition, and physiology between the pig and the human (Ball et al. 1996; Clouard et al. 2012). Pigs are also the only widely utilized animal model that is truly omnivorous, and they have strikingly similar nutritional requirements to that of humans (Patterson et al. 2008). The gut in the newborn pig is more mature than in newborn rodents, although less mature than in infants (Sangild 2006). Thus, in pigs, preterm delivery at 90 % gestation is comparable to preterm infants born at approximately 75 % gestation (30 weeks) (Siggers et al. 2011). In contrast to rodent models, the size of the newborn pig easily allows for clinically tissue collections and experimental manipulation of physiologic conditions. Besides, the ontogeny, the physiology of digestion, and associated metabolic processes are very similar between humans and pigs (Patterson et al. 2008; Patrycja and Barbara 2008), which makes the pig an attractive animal model for further studies on intestinal complications related to preterm birth.

3 Determinants in Intestinal Inflammatory Diseases

Preterm infants suffer from numerous devastating intestinal diseases, including necrotizing enterocolitis (NEC). Nutritional, microbial, and immunological dysfunctions may all play a role in disease progression. The lowered digestive and nutrient absorptive function, impaired intestinal epithelial barrier, inappropriate bacterial colonization, and a dysregulated mucosal immune system may add to this increased susceptibility to enteric disease.

3.1 Immature Digestion and Nutrient Absorption in Preterm Neonates

Preterm birth is associated with immature motility, digestive capacity, and nutrient absorption, thus leading to nutrient fermentation, bacterial overgrowth, and mucosal inflammation.

Gastrointestinal motility is limited largely to infants less than 34 weeks gestation (Riezzo et al. 2009). The motility is found considerably less organized in premature infants (Neu 2007), probably, because of the intrinsic immaturity of the enteric neurons. Also in preterm piglets, bowel movements are not well developed during the first days of enteral feeding (Sangild et al. 2002a, b, c). The incomplete innervation and poor motility of the immature gut, thereby, may lead to stasis, nutrient fermentation, inappropriate colonization, and further contribute to the development of enteric disease in preterm infants (Neu 2007; Oste et al. 2005).

Studies in both animals and infants indicate that immature brush-border enzyme activities following preterm delivery may result in maldigestion, excessive nutrient fermentation, intestinal distension, and mucosal ischemia in preterm infants. In preterm infants, the intestine is relatively short (Weaver et al. 1991) and may have a reduced absorptive area, consistent with studies in pigs (Sangild et al. 2000, 2002a, b, c). Besides, preterm-delivered pigs also differ from term neonates in their intestinal cell proliferative and apoptotic responses (Burrin et al. 2000; Bittrich et al. 2004). The impaired ability of the immature intestine to increase cell proliferation, decrease apoptosis, and regulate the mesenteric blood flow (Crissinger et al. 1994; Clark et al. 2005; Dyess et al. 1993) may lead to mucosal atrophy, dysfunction, and necrosis in preterm neonates. In addition to ontogenetic immaturity of enterocyte function, the possible hypoxia, hypothermia, altered endocrine and metabolic status may make preterm neonates more sensitive to serious feeding-induced complications (Sangild 2006).

These deficiencies in intestinal structure and function appear to be one of the most critical problems resulting in feeding intolerance, a commonly encountered problem in neonatal care. Formula-fed preterm neonates are thus at increased risk of developing diseases due to their compromised digestive system. Similar results are observed in preterm newborn pigs. Preterm birth affects the intestinal response to enteral nutrition in newborn piglets. Formula feeding in preterm newborn pigs leads to a diminished intestinal trophic responses relative to colostrum (Bjornvad et al. 2005; Oste et al. 2005; Sangild et al. 2006), marked atrophy of the mucosal surface, and increased permeability (Rouwet et al. 2002). Preterm neonates, thus, may require a period of total parenteral nutrition (TPN) before enteral nutrition is administered (Heird and Gomez 1996; Sangild et al. 2002a, b, c). Nevertheless, parenteral nutrition has significant detrimental side effects, including intestinal atrophy, malfunction, and sepsis leading to increased susceptibility to inflammatory stimuli and the development of intestinal inflammation (Siggers et al. 2011). This is consistent with the observation that TPN increases the risk of NEC in prematurely born piglets.

3.2 Deficient Host-Associated Defense Mechanisms in Preterm Neonates

Relative to term neonates, preterm neonates show immature intestinal barrier that lacks several key protective mechanisms that normally prevent invasion by luminal bacteria. The premature gastrointestinal tract also has increased intestinal permeability (Neu 1996) commonly observed in preterm neonates (Neu 2007), making the immature intestine more permeable to macromolecules, while little is known about the maturation of tight junction proteins such as occludin and claudins, which constitute the major paracellular barrier of the epithelium (Nusrat et al. 2000). The mucus layer forms a physical barrier between the underlying epithelium and the lumen of the gastrointestinal tract (Atumal et al. 2001), protecting the epithelium against noxious agents and pathogenic bacteria. However, the production of mucous was reported to be immature in preterm infants (Claud and Walker 2001; Omari and Davidson 2003; Sangild 2006), leading to a diminished intestinal barrier function, impaired mucosal repair, and lowered degradation of bacterial toxins.

Additionally, impaired functioning of immune defenses in preterms (Sangild et al. 2002a, b, c; Baxter 2010), as well as lower levels of immunoglobulins (e.g., IgA, IgM, IgG) (Lin 2004), gut B and T lymphocytes, makes premature neonates particularly susceptible to enteric inflammation and injury during the early postnatal period (Claud and Walker 2001; Kuitunen and Savilahti 1995). Coupled with the increased intestinal mucosal permeability in preterms (Rouwet et al. 2002; van Elburg et al. 2003), this impairment leads to transmural translocation of microbes or their toxic products into the immature intestinal mucosal barrier in neonates. This may, in turn, further compromise intestinal defense mechanisms and eventually culminate in an inflammatory cascade, leading to NEC (Berman and Moss 2011). Furthermore, the premature gastrointestinal tract also has decreased regenerative capabilities, and the imbalance between epithelial cell injury and repair usually leads to a vicious cycle of maldigestion, impaired mucosal protection, immune activation and results in a greater potential for tissue damage (Siggers et al. 2011).

3.3 Gut Microflora and Inflammatory Responses in Preterm Neonates

Several studies in both human infants and piglets show that assemblages of gut bacteria differ markedly between preterm and term neonates (Schmidt et al. 2008; Cilieborg et al. 2010). While the full-term infants are rapidly colonized with a more diverse microbiota, preterm neonates have a slow bacterial colonization and decreased bacterial diversity present, which may predispose the premature gut to bacterial overgrowth by pathogenic bacteria (Arboleya et al. 2012; Fanaro et al. 2003; Schwiertz et al. 2003), and this difference may be directly related to the degree of intestinal prematurity, the deficient mechanical defense barriers, as well as the environmental factors (Hallstrom et al. 2004). Wang et al (2011) showed that intestinal microbiota had important functions in host energy metabolism, amino acid nutrition, immunity, and health.

Moreover, the nature of the enteral foods may affect initial bacterial colonization patterns in preterm infants (Caicedo et al. 2005; Claud and Walker 2001) and pigs (Shulman 2002; Wang et al. 2011), and the use of parenteral nutrition further delay colonization (Fanaro et al. 2003; Caicedo et al. 2005) in neonates following preterm delivery. Maldigestion and disturbances in intestinal barrier function may lead to bacterial overgrowth and excessive nutrient fermentation, thereby rendering the mucosa more susceptible to bacterial infections and further initiating uncontrolled inflammatory reactions (Siggers et al. 2011).

A disordered enterocyte signaling to bacterial toxins, via the production of various pro-inflammatory cytokines (e.g., IL-1, IL-6, IL-8, TNF-alpha), is thought to be crucial in the development of NEC in the susceptible preterm infants (Hunter et al. 2008). Those pro-inflammatory mediators may further initiate the inflammatory cascade, thus favoring mucosal barrier disruption and adversely affecting mucosal repair. Toll-like receptors are identified among the immunological components of the early mucosal dysfunction. It has been shown that TLRs (2 and 4) are upregulated in intestinal tissue from preterm pigs with NEC (Sangild 2006). In addition, p38 kinase, cyclooxygenase-2, and NF-kB signaling pathways may all be involved in mucosal inflammation (Grishin et al. 2006; Wang et al. 2010). It remains difficult, however, to further understand why the premature newborn is susceptible to NEC as well as other inflammatory bowel diseases, which may need further study in preterm animal models.

4 Nutritional Modulation of Mucosal Defense and Immunology in Preterm Neonates

As previously stated, the immature neonates are prone to bacterial infection and exaggerated immune responses, potentially resulting in irreversible tissue damage. Mother’s milk and colostrum contains numerous bioactive factors, including growth factors, immunoglobulins, anti-inflammatory components, as well as amino acids (Møller et al. 2011; Claud et al. 2003), which help to protect against the development of intestinal diseases. Immunonutrition with specific nutrients may also be effective to modulate the activity of the immune system. Some dietary components, such as amino acids, have already been shown to enhance mucosal barrier function and immunologic responses in animal models and in humans (Stechmiller et al. 2004). Kim et al (2007) reported that provision of amino acids and fatty acids with specific functions may enhance the performance of pregnant and lactating sows by modulating key metabolic pathways by which can enhance conception rates, embryogenesis, blood flow, antioxidant activity, appetite, translation initiation for protein synthesis, immune cell proliferation, and intestinal development. Increasing evidence shows that dietary supplementation of specific amino acids to animals and humans with malnutrition and infectious disease enhances the immune status, thereby reducing morbidity and mortality (Li et al. 2007; Yin et al. 2010).

4.1 Arginine

The arginine, which is nutritionally essential for neonates, is involved in a number of biological and physiological processes. Arginine is crucial for the synthesis of protein and molecules (e.g., nitric oxide (NO), creatine, and polyamines) with enormous physiological importance (Flynn et al. 2002; Rhoads et al. 2004; He et al. 2009; Kim et al. 2007; Li et al. 2007; Wu et al. 2009). Nitric oxide is a vasodilator involved in intestinal permeability, mucosal integrity, and barrier function (Upperman et al. 2005; Wu et al. 2007). Moderate levels of NO are important for regulation of mesenteric blood flow and protect the mucosa from injury. Polyamines are involved in the regulation of gene expression, DNA and protein synthesis, apoptosis, as well as cellular division (Flynn et al. 2002). Moreover, arginine stimulates the secretion of growth hormone and insulin in preterm infants (Vlaardingerbroek et al. 2011), thereby playing an important role in regulating nutrients metabolism (Liu et al. 2008; Yao et al. 2008, 2011; Yin and Tan 2010; Tan et al. 2009, 2011).

Intestinal amino acid metabolism differs between preterm and term birth. A significant nutritional problem in preterm infants is a severe deficiency of arginine (hypoargininemia), which occurs in more than 50 % of the preterm infant population (Wu et al. 2004). Arginine deficiency may result in hyperammonemia as well as intestinal, immunological, and neurological dysfunction (Flynn et al. 2002) and it is often associated with an increased incidence of NEC in preterm infants (Becker et al. 2000). Thus, the knowledge of arginine metabolism and physiological effects is beneficial for optimizing neonatal survival and health in this compromised population.

Wu et al. (1999) reported that the amino acid composition of the fetal pig was similar to that of human fetus. It has been reported that endogenous synthesis of arginine is important for maintaining arginine homeostasis in the neonatal pigs (Flynn and Wu 1996), and the underdevelopment of intestinal arginine synthesis may be primarily responsible for hypoargininemia in preterm neonates (Dekaney et al. 2003). Glutamine and proline are major substrates for intestinal synthesis of citrulline in pigs (Wu et al. 2000a, b, 2009; Wang et al. 2008). However, synthesis of citrulline is low and there is little conversion of citrulline into arginine in enterocytes of preterm neonates owing to the limited expression of the genes for key enzymes (e.g., pyrroline-5-carboxylate synthase, argininosuccinate synthase, and lyase), thereby contributing to hypoargininemia (Wu et al. 2004). Furthermore, the possible increase in whole-body arginine catabolism, as well as absence of perinatal cortisol surge due to premature delivery, may also be responsible for the limited endogenous synthesis of arginine. In preterm piglets, low rates of intestinal arginine synthesis are associated with low plasma arginine concentrations (Urschel et al. 2007). Plasma levels of citrulline, arginine, and glutamine are lower in premature neonates with NEC compared with healthy infants (Wu et al. 2001; Becker et al. 2000); and provision of exogenous arginine prevents hyperammonemia and reduces NEC (Amin et al. 2002). Thus, an enhancement of endogenous arginine synthesis in preterm neonates may be obtained by the promotion of the maturation of intestinal arginine-synthetic enzymes.

Glucocorticoids play a crucial role in advancing the maturation of intestinal arginine synthesis and possibly decrease the incidence of NEC in preterm infants (Bauer et al. 1984). Administration of cortisol is effective to advance the maturation of intestinal arginine synthesis in preterm neonates (Wu et al. 2004). Another promising candidate is glucagon-like peptide-2 (GLP-2), a nutrient-responsive gut hormone, which may exert multiple effects on intestinal mucosa growth in preterm neonates (Estall and Drucker 2005). In neonates, proline is a dietary precursor for arginine and is dependent on intact gut metabolism (Vlaardingerbroek et al. 2011). Preterm infants receiving PN are unable to synthesize sufficient proline de novo (Miller et al. 1995). Future research is needed to define mechanisms for arginine metabolism and develop strategies for arginine deficiency in preterm infants.

Arginine plays an important role in improving intestinal function and regulating nutrient metabolism, but the underlying mechanisms are largely unknown (Liu et al. 2008). He et al. (2009) conducted metabolomic analysis of the response of growing pigs to dietary l-arginine and found that arginine alters the catabolism of fat and amino acids in the whole body, enhances protein synthesis in skeletal muscle, and modulates intestinal microbial metabolism. Tan et al (2011) indicated that Arg differentially regulates expression of fat-metabolic genes and increases mTOR signaling activity in skeletal muscle (Yao et al. 2008) and white adipose tissue, therefore favoring lipogenesis in muscle but lipolysis in adipose tissue.

4.2 Glutamine

Glutamine is the preferred fuel for rapidly proliferating cells including enterocytes (Chauhan et al. 2008). It is essential for many metabolic processes, and supplementation with this amino acid has been demonstrated to improve mucosal integrity and intestinal barrier function in critically ill patients. In vitro, glutamine is required for barrier function (Ewaschuk et al. 2011) and helps recovery from loss of transepithelial resistance and increase of permeability induced by stress in Caco-2 cells (Li et al. 2003). Wang et al (2008) found that early weaning resulted in increased expression of genes related to oxidative stress and immune activation but decreased expression of genes related to macronutrient metabolism and cell proliferation in the gut. Dietary glutamine supplementation increased intestinal expression (120–124 %) of genes that are necessary for cell growth and removal of oxidants, while reducing (34–75 %) expression of genes that promote oxidative stress and immune activation. Functionally, the glutamine treatment enhanced intestinal oxidative-defense capacity, prevented jejunal atrophy, and promoted small intestine growth and body weight gain in weaned piglets. These findings reveal coordinate alterations of gene expression in response to weaning and aid in providing molecular mechanisms for the beneficial effect of dietary glutamine supplementation to improve nutrition status in young mammals.

Glutamine is abundant in mother’s milk but present in much lower levels in formula milk (Agostoni et al. 2000). Thus, neonates, prematurely born infants, would benefit from glutamine addition. However, clinical studies of glutamine supplementation remain inconclusive. Parenteral glutamine appears to be well tolerated and safe in preterm neonates and this amino acid reduces the time to achieve full enteral nutrition (Thompson et al. 2003). Provision of glutamine showed some beneficial effects such as inhibition of whole body protein breakdown (Kadrofske et al. 2006) and activation of immune system in preterm infants (Parimi and Kalhan 2007). However, Tubman and Thompson (2001) reported that no additional benefit of the addition of glutamine to preterm infants was observed in their study. More studies are needed to evaluate the efficacy of this amino acid in neonatal nutrition and to understand the mechanism of glutamine dysfunction-related pathology.

4.3 Methionine and Cysteine

Methionine, an essential amino acid, is also a source through cysteine production for the synthesis of glutathione (GSH). This latter compound plays a crucial role in reducing intestinal oxidative damage and inflammation (Thomas et al. 2008). The rates of transsulfuration of methionine are high in prematurely born low birth weight infants (Maaike et al. 2007a, b). This may reflect high demands for glutathione (GSH) and methionine in parenteral amino acid mixtures for premature babies. It is reported that general cysteine requirement is less than 18 mg/kg per day and that cysteine is probably not a conditionally essential amino acid in the prematurely born infant (Maaike et al. 2007a, b). Regardless of the adequate GSH and protein synthesis, methionine has been implicated in increased homocysteine concentration (Courtney-Martin et al. 2008; Shoveller et al. 2004) in the neonate. Therefore, a balance between methionine and cysteine should be taken in consideration to provide the adequate total sulfur amino acid (SAA) in neonatal nutrition (Courtney-Martin et al. 2010).

4.4 Branched-Chain Amino Acids

The essential branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, are used for incorporation into body protein (Maingay-de Groof et al. 2010; Li et al. 2011), and utilization by intestine is also high. The uptakes of total leucine and valine carbon are relatively large and the oxidation rates of these essential branched-chain amino acids are high in fetuses and neonates (van den Akker et al. 2011). Among the BCAAs, leucine can act as a nutrient signal and stimulates protein synthesis via the activation of translation initiation factors (Vlaardingerbroek et al. 2011). Recent work with young pigs shows that reducing dietary protein intake can improve gut function after weaning but result in inadequate provision of essential amino acids for muscle growth. Yin et al. (2010) reported that supplementing l-leucine to a low-protein diet may maintain the activation of translation initiation factors and adequate protein synthesis in multiple organs of post-weaning pigs. This novel finding provides a molecular basis for designing effective nutritional means to increase the efficiency of nutrient utilization for protein accretion in neonates. Leucine is not only a substrate for protein synthesis of skeletal muscle but also plays as signaling molecules to affect feeding behavior, energy balance, and fuel efficiency (Li et al. 2011). Leucine activates signaling factor of mammalian target of rapamycin (mTOR) to promote protein synthesis in skeletal muscle and in adipose tissue. It is also a major regulator of the mTOR sensitive response of food intake to high protein diet. Meanwhile, leucine regulates blood glucose level by promoting gluconeogenesis and aids in the retention of lean mass in a hypocaloric state. It is beneficial to animal nutrition and clinical application and extrapolation to humans (Li et al. 2011). Studies in newborn pigs suggest that enteral leucine supplementation may have a beneficial effect on neonatal growth as it may enhance protein synthesis in an mTORC1-dependent pathway (Suryawan et al. 2012; Li et al. 2011). The gut has a high demand for leucine and protein synthesis has been found to be limited by deficient leucine intake (Elango et al. 2002). BCAA-enriched parenteral nutrition in preterm neonates might influence functional outcome in the direct postnatal phase. The requirements of the individual BCAAs are almost twice the current recommendations (Maingay-de Groof et al. 2010). To optimize current parenteral and enteral feeding, the optimal BCAA ratio should be determined for both. Further studies are required to better understand the role of BCAAs in the regulation of neonatal growth.

4.5 Threonine

Threonine is an indispensable amino acid that must come from dietary sources. It is critical in the production of mucins in the gut (Schaart et al. 2009). Therefore, this amino acid is of presumably vital nutritional importance to maintain the protective mucus layer and thus the intestinal barrier function. Lack of threonine can result in diarrhea and reduced mucin production, indicating the important role of threonine in the structure and function of the gut (Vlaardingerbroek et al. 2011). However, prolonged dietary excess of threonine fed to neonates, may have negative behavioral consequences and may induce serious metabolic disturbances (Chapman et al. 2009). Dietary threonine imbalance is known to reduce the growth of the small intestine, liver, and skeletal muscle in young animals. Using the pig model, Wang et al. (2007) found that either a deficiency or an excess of dietary threonine impairs protein synthesis in these tissues. This finding provides a mechanism for the low growth performance of animals fed a threonine-imbalanced diet. Currently, neonatal amino acid solutions provide intakes of threonine (111–165 mg/kg per day) are greater than an infant’s enteral intake from breast milk (76 mg/kg per day) (WHO 2007). Chapman et al. (2009) concluded that current parenteral solutions should be revised to incorporate the population-safe requirements of threonine to promote optimum metabolic and neurologic growth in neonates.

4.6 Tryptophan

Tryptophan is an essential amino acid while the concentration is low in plasma and content low in proteins compared with the other essential amino acids (Vlaardingerbroek et al. 2011). Intestinal inflammation, malnutrition, and pro-inflammatory situation may result in tryptophan depletion (Christmas et al. 2011), thus affecting the weight gain and nitrogen balance in neonates. In young pigs, experimental inflammation was associated to a decrease in plasma tryptophan concentrations compared with healthy piglets (Le Floc’h et al. 2008). Furthermore, studies showed that in neonatal piglets, no difference is found in tryptophan requirements when enteral and parenteral feeding are compared (Alegria et al. 1999). Therefore, most parenteral neonatal amino acid solutions contain similar concentrations of tryptophan compared with human breast milk (Cvitkovic et al. 2004). Lastly, the commercially available preterm formula content ranges from 18 to 36 mg/kg per day when infants receive 160 ml/kg per day of milk (Vlaardingerbroek et al. 2011).

4.7 Other Amino Acids

Taurine is considered conditionally essential because needs are not met when intake is low (Verner et al. 2007). It has important roles in intestinal absorption, membrane stability, and visual development in preterm infants. Currently, taurine concentrations in modern parenteral amino acid solutions are more than sufficient to meet recommendations (Verner et al. 2007). Tyrosine is also a conditionally essential amino acid resulting from the insufficient enzymatic activity in preterm infants. Due to poor tyrosine solubility in parenteral nutrition (Roberts et al. 2001), hydroxylation of phenylalanine to tyrosine may be a good way to provide tyrosine when the diet is tyrosine-deficient.

5 Conclusion

Deficiencies in intestinal integrity, barrier function, digestive capacities, and intestinal immunity, make preterm neonates more susceptible to inflammatory diseases. Using preterm pigs, recent evidence suggests that nutritional modulation has great potential to improve neonatal intestinal development, manipulate the gut microbiota, in parallel with direct effects on the mucosal immune system, to prevent the onset of NEC. Some amino acids have been shown to enhance mucosal barrier function, immunologic responses, and NEC resistance in piglets and in neonates. However, it always remains a challenge to translate data generated from animal models to corresponding conditions in humans. Understanding the unique function of amino acids could eventually play a pivotal role in improving future nutritional strategies for premature infants. Clearly, further studies involving amino acids as compounds for prevention and clinical therapies against devastating intestinal diseases are needed, both in parenterally and enterally fed infants. Such work will greatly advance our knowledge with regard to the “optimal” amino acid pattern and it will also be beneficial for designing the next generation of amino acid supplemental solutions to optimize survival and health in preterm neonates.