Lipids and fatty acids are critical for optimal cell physiology, organ function, and neurodevelopment. Although these nutrients are delivered to the preterm infant, specific considerations and challenges unique to this population have not been overcome to efficiently extract the physiologic and developmental benefits conferred by these bioactive factors. Some of these factors include understanding the ideal substrate, target dosing, hydrolysis and absorption, and metabolic conversion.
This chapter will discuss the challenges that limit optimal lipid and fatty acid delivery in the preterm infant. Potential strategies to overcome these limitations will be discussed across the spectrum of parenteral to enteral phases of nutrition. Lessons will be drawn from another vulnerable population that share impairments in lipid and fatty acid processing; namely, patients with cystic fibrosis.
Lipids and Fatty Acids Maintaining Function and Health
Thousands of lipid species across six major categories have been detected circulating in the plasma and integrated in cellular membranes. Lipids play critical roles in maintaining function and health throughout an individual’s life span and provide an energy-rich source of nutrition supporting normal cellular functions and somatic growth. Lipids form the structure of human cellular and neural membranes vital for cell-to-cell communication and downstream signaling to carry out critical physiologic functions, including organogenesis, immune function, and regulation of inflammation. Fatty acids are a major building block of these lipids and are central to mediating these biologic processes.
To ensure that preterm infants have sufficient nutritional pools of lipids and fatty acids to draw upon and recruit for these critical biologic processes, several key steps must be accomplished: (1) determining the nutritional requirements of lipids and fatty acids to sustain these biologic processes during the postnatal transition after an abrupt discontinuation of maternal supply; (2) employing delivery strategies that span across the parenteral and enteral phases of nutrition; and (3) overcoming the developmental insufficiencies in lipid and fatty acid hydrolysis, absorption, and metabolism. When these steps are unrealized, the accretion of critical lipids and fatty acids is compromised. Equally important, when the metabolism of these nutrients is neither mature nor supported, abnormal generation and accumulation of intermediate metabolites and oxidized byproducts may occur, potentially causing harm.
Several recent reviews have summarized our current understanding of lipid and fatty acid requirements for the preterm infant. However, what remains unclear is how to best achieve optimal lipid and fatty acid delivery. How do we best support these requirements during the parenteral and enteral phases of nutrition? This chapter will review each phase of postnatal nutrition and the unique challenges faced by the preterm infant and describe potential strategies that may overcome these obstacles. To address these issues, we will reflect on other populations with fatty acid alterations, largely cystic fibrosis, and draw upon potential parallels in shared physiology that may inform future nutritional strategies in the preterm infant.
Mechanisms and Significance of Fatty Acid Alterations in Cystic Fibrosis
Essential fatty acid deficiency (EFAD), rarely seen in developed countries, is caused by the inadequate consumption in the diet of alpha linolenic acid (18:3, n-3) and gamma linoleic acid (18:2, n-6). These two fatty acids are essential because they cannot be endogenously synthesized and rather must be sourced from the diet. They are also the precursors to the biosynthesis of downstream fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA). EFAD can present as dermatitis, thrombocytopenia, and growth failure. The major biochemical changes reflective of EFAD are decreased AA and increased mead acid, the latter being a downstream product of oleic acid, an omega-9 fatty acid. Since cells need to maintain a constant number of double bonds to preserve membrane fluidity and mechanics, EFAD is associated with and diagnosed by a plasma triene/tetraene ratio >0.2. The elevated triene/tetraene ratio in patients with cystic fibrosis suggests the presence of a biochemical EFAD and has opened the investigation of altered fatty acid metabolism in this population.
Defects in Fatty Acid Metabolism
Cystic fibrosis (CF) is a multiorgan disease associated with inspissated secretions in the sinuses, lungs, intestine, biliary and pancreatic ducts, and male reproductive tract. Despite identification of the gene responsible for CF as the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel, many questions remain as to how this defect leads to the myriad of cellular and host abnormalities seen in CF. This includes the excessive host inflammatory response and viscous secretions with abnormal mucin composition.
The authors’ group has been focused on determining whether alterations in fatty acid metabolism may lead to the excessive host inflammatory response in CF, even in the absence of infection. It was first determined that in the pancreas and lung from UNC exon 10 CF knockout mice, compared with wild-type littermates, there was a twofold decrease in membrane-bound DHA and a reciprocal twofold increase in membrane bound AA. Linoleic acid levels were also low. These fatty acid abnormalities were seen only in CF-affected organs and represent the first disease linked to alterations in membrane-bound AA.
Feeding these mice with DHA, 40 mg/day for 7 days, led to normalization of the fatty acid defect and reversal of the CF pathology in the pancreas and ileum. Although there is no spontaneous lung disease in these CF knockout mice, administration of aerosolized Pseudomonas lipopolysaccharide once a day for 3 days resulted in an exaggerated host inflammatory response based on bronchoalveolar lavage showing 4 million/mL neutrophils from CF knockout mice compared with 1.5 million/mL in wild-type littermates. Oral DHA, 40 mg/day for 7 days, normalized neutrophil counts in the CF knockout mice to wild-type levels, but DHA had no effect on further reducing neutrophil levels when fed to wild-type mice. The mechanism of action of DHA was through reductions in the downstream eicosanoid metabolites PGF-2α and PGE2, with no effect on tumor necrosis factor and the murine interleukin-8 equivalents MIP2 and KC. The altered fatty acid imbalance was also linked to decreased expression of peroxisome proliferator-activated receptor (PPAR)-[gamma].
Subsequent studies in humans with CF have demonstrated a similar fatty acid abnormality, which is directly correlated with the degree of CFTR dysfunction with progressive alterations seen when progressing from heterozygotes to those with mild CFTR mutations to those with severe CFTR mutations. This was unrelated to age, body mass index, or comorbidities. Furthermore, these fatty acid alterations were not seen in other non-CF inflammatory diseases but, rather, are inherently linked to CFTR dysfunction.
The low linoleic acid levels that have been described in several studies of patients with CF were thought to be reflective of an EFAD despite sufficient linoleic acid in the diet. In airway cells in culture, with or without functional CFTR, by using sense and antisense messenger RNA (mRNA) strategies, it has been observed that the low linoleic acid levels are caused by increased conversion to AA through upregulation of the delta 6 desaturase enzyme. Normally this is the rate limiting step in the metabolism of linoleic acid to AA but with loss of normal CFTR function, the “brakes” on this pathway are lost. It remains unknown whether the low DHA levels result from a primary decrease in the biosynthesis of DHA or are secondary to increased AA levels. There are ongoing studies examining whether strategies to increase DHA levels have an impact on CF related disease.
Pancreatic Insufficiency and Lipid Maldigestion
In addition to altered fatty acid metabolism in CF, exocrine pancreatic insufficiency is present in approximately 85% of patients. This inflammatory and fibrosing process begins in utero with full expression of exocrine pancreatic insufficiency typically by the end of the first year of life. Although carbohydrate and protein malabsorption occurs, it is the fat maldigestion that is responsible for most symptoms, including steatorrhea, bloating, and abdominal distention with gas. Typical coefficients of fat absorption, referred to as coefficient of fat absorption (CFA) , range from 20% to 70%. Before the advent of pancreatic enzyme replacement therapy, babies with CF died within 1 year as a result of profound protein and fat malnutrition. Institution of pancreatic enzymes has truly been lifesaving in this disease.
Defects in Intestinal Absorption
Not only is there exocrine pancreatic insufficiency resulting in maldigestion in CF, but there is also intestinal malabsorption. Thus whatever hydrolysis of fats occurs as a result of native or exogenous pancreatic enzymes, there is variable impairment in nutrient absorption across the intestinal epithelial cells caused by defective CFTR function. Other mechanisms to explain this malabsorption include thick mucus resulting in a greater unstirred intestinal surface layer; decreased luminal pH affecting pancreatic enzyme function, which has an optimal pH above 6.5, as well as precipitation of bile salts; quantitative and qualitative changes in bile composition; changes in the intestinal microbiome; and alterations in nutrient transporters. Taken together with exocrine pancreatic insufficiency, this combination of maldigestion and malabsorption has a major impact on nutritional status. The importance of nutritional status in CF cannot be overemphasized and is inextricably linked to lung disease. For example, weight-for-age percentile at age 4 years has been shown to be predictive of forced expiratory volume in one second at age 18 years in patients with CF.
Role of Ileal Brake in Feeding Tolerance
Dysmotility is present in at least a third of patients with CF and includes gastroparesis as well as small and large intestinal involvement. The mechanisms are diverse but appear to be a result of altered CFTR function in intestinal ganglia, as well as altered luminal pH and dysbiosis. Diabetes mellitus is present in up to 50% of patients with CF by the time they reach adulthood and can also impact gut function directly by affecting gut innervation and indirectly by altered microbiome and transporters. Furthermore, in the approximately 15% of patients with CF requiring nocturnal enteral tube feeds because of malnutrition, it is not unusual for patients to awaken and display all the signs and symptoms of ileal brake activation due to undigested and/or poorly absorbed full-length triglycerides or free fatty acids.
Each of the mechanisms leading to fatty acid alterations in CF have a potential role in the physiology of lipid digestion and fatty acid balance in the preterm infant—impaired metabolism, pancreatic insufficiency, impaired intestinal absorption, and the ileal brake. Successful strategies to optimize lipid and fatty acid delivery need to consider these facets and within developmental context as the preterm infant transitions from in utero nutrition to receiving parenteral nutrition and finally to the time of full enteral nutrition.
Lipid and Fatty Acids in Preterm Infants
With limited time in utero to establish nutritional stores, the preterm infant is highly vulnerable to quickly acquiring postnatal deficits in essential macronutrients as well as their building blocks that serve as important immuno-nutrients for the developing infant. Parenteral nutrition remains the first bridge to minimize these nutritional deficits. The precise postnatal fatty acid requirements of the preterm infant have not been fully established; however, if the goal is to maintain levels that would otherwise be seen in utero, the currently available lipid emulsions fall short of this goal.
Soybean oil lipid emulsions (SOLEs) remain the predominant lipid emulsion used in preterm infants in North America. SOLEs provide the essential fatty acids of linoleic acid (LA, 18:2n6) and alpha-linolenic acid (ALA, 18:3n3) but little to none of the downstream long-chain polyunsaturated fatty acids. Preterm infants exposed to SOLE demonstrate a twofold to threefold reduction in blood levels of DHA (22:6n3) and AA (20:4n6) concomitant with a 2.5-fold increase in LA. Thus the absolute levels of these fatty acids within the first postnatal week, as well as the ratios to one another, are reversed relative to profiles present in utero. Numerous health consequences have been described to be associated with these excesses and deficiencies of systemic fatty acid levels. Clinically, lower DHA levels have been linked to impaired neurodevelopment, retinopathy, and chronic lung disease, whereas low AA has been linked to late-onset sepsis. Preclinical data support the role of fatty acids and their terminal mediators in brain and eye health, pulmonary development, immune function, and intestinal development, including microbial colonization. Thus it is critical to understand postnatal fatty acid requirements and how best to deliver these bioactive nutrients before the onset of deficits.
Next-generation lipid emulsions that include various amounts of fish oil, which, compared with SOLE, provide greater concentrations of DHA and EPA (20:4n6), a small amount of AA, and little essential fatty acids (LA and ALA), have been developed. Concentrated fish oil-based lipid emulsions have been shown to alleviate the effects of traumatic brain injury in older children and adults, and this speaks to the potent bioactive properties of these omega-3 polyunsaturated fatty acids. Additionally, in infants, 100% fish oil lipid emulsions (FOLEs) have shown tremendous promise as a therapeutic strategy for parenteral nutrition associated liver disease.
The use of 10% to 15% FOLE has been studied as maintenance therapy in small cohorts of preterm infants with desirable trends in the incidence of bronchopulmonary dysplasia, retinopathy of prematurity, and cholestasis. However, of cautionary note, untoward alterations in fatty acid profiles and lipid metabolism are seen with the use of fish oil-based lipid emulsions. As expected, there is an increase in DHA levels when using FOLEs versus SOLEs, but this increase does not eliminate the early postnatal decline in DHA. Additionally, there is a substantial increase in EPA levels. The health effects of an increase in EPA are unknown; however, the well-established role of EPA in inhibiting platelet aggregation is a theoretical concern in the preterm infant. The rise in total omega-3 levels leads to a compensatory reduction in AA even lower than levels observed with SOLEs. AA is critical for infant growth and the reduction in systemic AA levels has been linked with an increase in nosocomial sepsis. Thus it is critical to develop a parenteral lipid emulsion that at least minimizes further reductions in AA and preferably maintains birth levels.
The use of FOLE may have other metabolic consequences. An analysis of lipid profiles between two comparable lipid emulsions except for the fish oil content (0% versus 10% fish oil) at a maximum dose of 2.6 to 2.8 g/kg/day revealed reduced lipogenesis in the fish oil group with reduced free cholesterol, cholesterol esters, and phospholipids—all complex lipids essential for organ development. In contrast, preterm infants who received FOLE at a dose of 3.5 g/kg/day versus a SOLE-based product at 2.5 to 3.5 g/kg/day demonstrated increased phospholipid, triglyceride, and free cholesterol levels. The authors suggest that this profile reflects lipid intolerance. However, with the increased need for lipid substrates in organogenesis, it is unclear what the ideal lipid profile is in a rapidly growing preterm infant. In addition, this latter study is confounded by the other compositional differences between the two lipid emulsions. Further studies are needed to understand the precise dose response impact of fatty acid delivery on lipid metabolism. Given the diverse role of complex lipids in human physiology, a sacrifice of one class of lipids for another will unlikely be tolerated well.
In summary, the currently available parenteral SOLEs and FOLEs fail to meet the unique lipid and fatty acid needs of the preterm infant. Ideally, lipid emulsions need to maintain birth levels and mitigate the changes in the systemic fatty acids levels currently observed after delivery. Although FOLEs minimize the postnatal DHA deficit, they induce further declines in AA and considerable increases in EPA. Additionally, as the omega-3 to omega-6 balance changes, it is expected that other complex lipid profiles will be altered as well. In adults, some of these changes, such as decreasing triglyceride levels in hypertriglyceridemia or reducing adipokines, including leptin, are desirable to promote weight loss. However, reduced lipogenesis and alterations of adipokines may not be desirable for preterm infants who rely on these compounds for organ growth and development. The long-term effects of these induced trade-offs with manipulations in the omega-3 to omega-6 balance need to be investigated thoroughly before wide spread adoption in the routine use of FOLEs.
Preterm infants receive small, steady advancements in enteral nutrition reaching full-volume enteral feedings at an approximate median age of 14 postnatal days. With complete transition from parenteral to enteral nutrition, all lipid and fatty acid requirements must now be met by the enteral diet alone. However, maintaining fatty acid levels close to those seen in utero cannot be achieved enterally using current feeding strategies and available nutritional substrates (human milk, human milk fortifiers, and/or preterm formula). Several factors have led to this conclusion. First, the pace of increasing enteral feeding volumes cannot overcome the deficit in DHA and AA seen within the first postnatal week when the infant is largely dependent on parenteral nutrition. Second, the fatty acid content in human milk, fortifiers, and formula is not sufficient to restore systemic fatty acid levels. Third, preterm infants express a developmentally immature digestive capability for efficient lipid hydrolysis and absorption of fatty acids when fed triglyceride oils. The latter is mediated by developmental exocrine pancreatic insufficiency of the newborn.
Developmental Exocrine Pancreatic Insufficiency
Although not commonly recognized, infants are the largest population of exocrine pancreatic insufficient individuals. In 1980 Lebenthal and Lee conducted a study in which they placed an oral–duodenal tube in healthy term infants and measured amylase, trypsin, and lipase secretion in response to cholecystokinin and secretin stimulation. Up to the first 6 months of life, protease secretion was near adult levels, but there was little amylase and lipase secretion. The lack of the latter two enzymes is compensated for by the presence of amylase and bile salt stimulated lipase in breast milk. However, in formula-fed or donor milk-fed infants, the absence of significant levels of lipase and amylase would be expected to result in fat and carbohydrate malabsorption, respectively.
Fat Maldigestion and Specific Fatty Acid Absorption Coefficients
Humans possess lingual and gastric lipases that assist with fat digestion. Gastric lipase has preference for cleavage at the sn -3 position, which favors short- and medium-chain triglycerides, in contrast to pancreatic lipase, which preferentially hydrolyzes at the sn -2 position of long-chain triglycerides. Although gastric digestion was initially thought to be higher in preterm infants now it is thought to be similar to adults, where gastric lipase is responsible for 10% to 30% hydrolysis of ingested triglycerides. Thus the remainder of fat digestion is largely dependent on pancreatic lipase and the lower gastrointestinal tract.
CFA measurements in term and preterm infants demonstrate the impaired ability of formula-fed infants to digest fat compared with breast milk-fed infants. The authors’ group evaluated the coefficients of absorption for specific fatty acids in the diet for a cohort of preterm infant fed with mother’s milk or preterm formula. A 3-day collection of feeding and fecal samples allowed for gas chromatography–mass spectrometry quantification of fatty acids entering the body and fatty acids lost in the fecal output and thus not absorbed. An analysis was done at postnatal age 2 and 6 weeks. The authors found that absorption coefficients in preterm infants fed mother’s milk were sufficient at >90% for most fatty acids at both time points. In contrast, in formula-fed infants at both time points, a decrease in absorption coefficients was found with saturated and polyunsaturated fatty acids >12 carbons in length. The greatest impairment in fatty acid uptake was seen with DHA, where formula-fed infants were less efficient compared with breast milk-fed infants at postnatal age 2 and 6 weeks (83.4% versus 96.2% and 74.9% versus 97.4%, respectively). Although it has been determined that enteral feeding matures the gut and promotes enzyme maturation and digestive abilities, the fact that at postnatal age 6 weeks, differences were still observed in fatty acid absorption by group, suggests that the dietary substrate is critical to these processes. Additionally, this may also explain why after the initial deficit in AA and DHA in the first postnatal week, enteral diet alone is unlikely to close this deficit. This delayed maturation in lipid digestion must be taken into account when considering approaches to lipid and fatty acid supplementation. It is important to note that deficits in long-chain fatty acid absorption were evident in the authors’ study, even though the total fat absorption between these two groups did not differ. Thus future studies in lipid digestion in preterm infants should distinguish between lipid classes and molecules for a more complete assessment of digestive capabilities.
In severe fat maldigestion independent of etiology, impairments in growth and absorption of other nutrients, such as fat-soluble vitamins, are observed. Additionally, pancreatic steatorrhea can present with bloating, abdominal distention, and oily, foul-smelling bulky stools. These symptoms are most evident in adult and pediatric populations with severe pancreatic insufficiency, such as CF, chronic pancreatitis, pancreatic cancer, and Shwachman–Diamond syndrome. Although we rarely ascribe severe fat maldigestion to preterm infants, some of the clinical presentations of feeding intolerance perhaps can be attributed to this impaired process (abdominal distension; loose, foul-smelling stools; and poor growth).
Bioavailability of Other Lipid Classes
Unique to human milk is the delivery of fat as milk fat globules (MFGs). Human MFGs have a triglyceride hydrophobic core surrounded by multiple layers of proteins, enzymes, and other complex lipids, such as phospholipids and cholesterol. Sphingolipids represent an important class of lipids embedded in these membranes, of which sphingomyelin is the most prevalent at 40% of the total MFG polar lipids. Sphingomyelin and the downstream bioactive metabolites had been shown to have beneficial properties for neonatal gut development as well as immune ontogeny. Interestingly, commensal gut microbiota also produce sphingolipid molecules that have been shown to be critical in the early postnatal period for gut and immune development, further supporting the bioactive role of these compounds.
Lipid digestion of the MFG membrane appears to be independent of pancreatic enzymes (in contrast to the triglycerides present in the core of the fat globule). Sphingomyelin is hydrolyzed from the MFG membrane upon contact with the intestinal brush border by the enzyme alkaline sphingomyelinase, also known as nucleotide phosphodiesterase pyrophosphatase 7 . Meconium analysis has shown that this enzyme is present at birth for both preterm and term infants. The activity of these enzymes, however, has not been completely defined. Additionally, hydrolysis of sphingomyelin is bile salt dependent, which is limited in the enteral circulation of preterm infants. It would be of interest to further characterize the gestational age dependency of MFG hydrolysis. A parallel concept exists for protein digestion. Although peptidases are present along the preterm intestinal brush border for hydrolysis of small peptide fragments, the pancreatic enzymes that initially hydrolyze the larger intact proteins are deficient. Thus despite the presence of brush border enzymes, the overall efficiency of protein digestion is diminished in preterm infants compared with more mature systems.
Other factors that might dampen the appropriate processing of MFG in the preterm infant are the effects of human milk handling. Takahashi et al. demonstrated that freezing human milk enlarges the diameter of the milk fat globule. In addition, within 12 hours of human milk fortification with a standard human milk fortifier, the milk fat globule size is increased with a decrease in the total surface area per unit of mass. The impact of this on the efficiency of fat digestion is not known.
Complex lipids, such as sphingomyelin, that reside in the membrane of human MFGs are important signaling factors for gut and immune development. To advance the immuno-nutrient potential of the dietary substrates presented to preterm infants, investigation of digestive capabilities and modifiers to this efficiency should be investigated further.
Ileal Brake and Feeding Intolerance
Intestinal digestion, absorption, and motility are tightly controlled to optimize nutrient processing. A feedback loop or network between nutrient presentation, sensing cells, and gut hormone production sets the pace of intestinal motility after a meal. When activated, this feedback system slows gut transit time (“brake”) and suppresses appetite to allow appropriate processing time of the meal. Although these “brakes” are in the jejunum and ileum, the most robust feedback system is within the distal ileum. Digested and undigested lipids are the most potent activators of this system, although undigested carbohydrates present in the ileum can also trigger this feedback loop. The greatest inhibition is seen with free fatty acids compared with intact triglycerides. Regarding chain length, long-chain fatty acids are more potent at activating the ileal brake with intermediate responses seen with medium-chain triglycerides. There are multiple signaling pathways that activate the ileal brake. These include glucagon-like peptide 1 (GLP-1) and peptide YY (PYY), which are synthesized and secreted locally within the L cells of the ileum. GLP-1 is also secreted from the pancreatic islets. In addition to inhibition of ileal motility, these two peptides inhibit gastric emptying, and both gastric and pancreatic secretion. Neural pathways, including vagal stimulation, have also been implicated in the ileal brake phenomenon.
In CF, activation of the ileal brake mechanism resulting from the presence of undigested fats and/or malabsorption of free fatty acids or carbohydrates may explain the symptoms of early satiety, bloating, and abdominal distention when continuous nocturnal feedings are delivered through an enteral tube without the benefit of pancreatic enzymes. No formal studies of the ileal brake system have been conducted in the preterm infant. However, not unlike patients with CF, the undigested lipid load reaching the distal ileum can be significant enough to prolong transit time, which can be up to 96 hours. The few studies of gut hormone responses after feeding do suggest an intact GLP-1 and PYY response making it conceivable that the ileal brake in preterm infants may be a factor determining feeding tolerance.