Anders D. Andersen

Molecular fingerprints of the human fecal microbiota from 9 to 18 months old and the effect of fish oil supplementation.

Objectives: The aim of this study was to monitor changes in the fecal microbiota from 9 to 18 months and to investigate the effect of increasing dietary n-3 polyunsaturated fatty acids on the fecal microbiota. Patients and Methods: In a double-blind controlled trial with random allocation to daily supplementation with 5 mL of fish oil (FO) or sunflower oil (SO) from 9 to 18 months of age, stool samples were collected from 132 healthy Danish infants. Molecular fingerprints of the bacterial DNA were obtained by terminal restriction fragment length polymorphism (T-RFLP). Results: The T-RFLP profiles indicated that a few T-RFs became dominant with age (bp100 and 102, both presumed to be Bacteroidetes) concomitantly with an overall increase in the microbial diversity (P = 0.04). Breast-feeding influenced both the T-RFLP profiles at 9 months and the changes from 9 to 18 months, and breast-feeding cessation during the trial modified the response to the dietary oils. In the FO group, the increase in bp102 was significantly reduced among children weaned before compared with those weaned during the trial (P = 0.027), whereas the increase in bp100 was reduced in the preweaned children of the SO group relative to those weaned during the trial (P = 0.004). This was supported by intervention group differences in the changes in bp102 and bp100 among the earlier weaned children (P = 0.06 and P = 0.09, respectively). Conclusions: Cessation of breast-feeding played a dominant role relative to developmental changes in the fecal microbiota from 9 to 18 months. FO compared with SO supplementation affected changes in large bacterial groups, but only among children who had stopped breast-feeding before 9 months of age.

A randomized controlled intervention with fish oil versus sunflower oil from 9 to 18 months of age: exploring changes in growth and skinfold thicknesses

n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA), from fish oil (FO), in rodents have been shown to reduce adipogenesis. Evidence of an effect on adipose tissue mass in humans is limited, and no studies have specifically aimed to elucidate this in infancy. To explore whether n-3 LCPUFA intake affects adipose tissue growth, we randomly allocated 154 healthy infants to daily supplementation with FO or sunflower oil (SO) from 9 to 18 mo of age and measured z-score changes in various anthropometric assessments of body size and skinfold thicknesses and plasma adipokine concentrations. Among the 133 completing infants, erythrocyte n-3 PUFA increased more in those receiving FO than in infants receiving SO [12.2 ± 0.7 (mean ± SE) versus 2.0 ± 0.4 fatty acid percentage (FA%), p < 0.001] with a concomitant larger decrease in n-6 PUFA (−8.9 ± 0.7 versus −0.9 ± 0.6 FA%, p < 0.001). We found no association between FO consumption relative to SO consumption and any of the anthropometric measures related to the size of the fat mass, but infants in the FO group had a lower skinfold ratio (triceps/subscapular) at 18 mo than those in SO group (p = 0.02). Our findings do not support the hypothesis that dietary n-3 LCPUFA is important for infant fat mass, but future studies testing this specifically are warranted.

Delayed growth, motor function and learning in preterm pigs during early postnatal life

Preterm birth interrupts normal fetal growth with consequences for postnatal growth and organ development. In preterm infants, many physiological deficits adapt and disappear with advancing postnatal age, but some may persist into childhood. We hypothesized that preterm birth would induce impaired organ growth and function during the first postnatal week in pigs, while motor abilities and behavioral characteristics would show more persistent developmental delay. Cesarean-delivered preterm (n = 112, 90% gestation) or term (n = 56, 100% gestation) piglets were reared under identical conditions and euthanized for blood and organ collection on postnatal days 0, 5, or 26. Body weight gain remained lower in preterm vs. term pigs up to day 26 (25.5 ± 1.5 vs. 31.0 ± 0.5 g·kg(-1)·day(-1), P < 0.01) when relative weights were higher for brain and kidneys and reduced for liver and spleen. Neonatal preterm pigs had reduced values for blood pH, Po2, glucose, lactate, hematocrit, and cortisol, but at day 26, most values were normalized, although plasma serotonin and IGF 1 levels remained reduced. Preterm pigs showed delayed neonatal arousal and impaired physical activity, coordination, exploration, and learning, relative to term pigs (all P < 0.05). Supplementation of parenteral nutrition during the first 5 days with an enteral milk diet did not affect later outcomes. In preterm pigs, many physiological characteristics of immaturity disappeared by 4 wk, while some neurodevelopmental deficits remained. The preterm pig is a relevant animal model to study early dietary and pharmacological interventions that support postnatal maturation and neurodevelopment in preterm infants.

Dietary long-chain n-3 PUFA, gut microbiota and fat mass in early postnatal piglet development—exploring a potential interplay

Dietary n-3PUFA and gut bacteria, particularly Bacteroidetes, have been suggested to be related to adiposity. We investigated if n-3PUFA affected fat storage and cecal bacteria in piglets. Twenty-four 4-day-old piglets were allocated to formula rich in n-3PUFA (∼3E%) from fish oil (FO) or n-6PUFA from sunflower oil (SO) for 14 days. We assessed body weight, fat accumulation by dual-energy X-ray absorptiometry and microbial molecular fingerprints. Dietary PUFA-composition was reflected in higher erythrocyte n-3PUFA in the FO- than the SO-group (P<0.001). Principal component analysis revealed group differences in the overall microbiotic composition, which involved a larger Bacteroides community in the SO-group (P=0.02). There was no significant difference in body fat percentage and no relationship between fat accumulation and gut Bacteroides. Hence, this study does not support an impact of n-3PUFA or microbiota on fat accumulation during the postnatal maturation period. The impact of dietary PUFA on the gut Bacteroides warrants further investigation.

Rapid gut growth but persistent delay in digestive function in the postnatal period of preterm pigs

Preterm infants often tolerate full enteral nutrition a few weeks after birth but it is not known how this is related to gut maturation. Using pigs as models, we hypothesized that intestinal structure and digestive function are similar in preterm and term individuals at 3-4 wk after birth and that early enteral nutrition promotes maturation. Preterm or term cesarean-delivered pigs were fed total parenteral nutrition, or partial enteral nutrition [Enteral (Ent), 16-64 ml·kg(-1)·day(-1) of bovine colostrum] for 5 days, followed by full enteral milk feeding until day 26 The intestine was collected for histological and biochemical analyses at days 0, 5, and 26 (n = 8-12 in each of 10 treatment groups). Intestinal weight (relative to body weight) was reduced in preterm pigs at 0-5 days but ENT feeding stimulated the mucosal volume and peptidase activities. Relative to term pigs, mucosal volume remained reduced in preterm pigs until 26 days although plasma glucagon-like peptide 2 (GLP-2) and glucose-dependent insulin-trophic peptide (GIP) levels were increased. Preterm pigs also showed reduced hexose absorptive capacity and brush-border enzyme (sucrase, maltase) activities at 26 days, relative to term pigs. Intestinal structure shows a remarkable growth adaptation in the first week after preterm birth, especially with enteral nutrition, whereas some digestive functions remain immature until at least 3-4 wk. It is important to identify feeding regimens that stimulate intestinal maturation in the postnatal period of preterm infants because some intestinal functions may show long-term developmental delay.

Physical activity level is impaired and diet dependent in preterm newborn pigs.

Background:Preterm infants show delayed development of motor function after birth. This may relate to functional immaturity of many organs, including the gut and brain. Using pigs as model for preterm infants, we hypothesized that early initiation of enteral feeding stimulates both gut growth and neonatal physical activity.Methods:In experiment 1, preterm and term pigs were fed parenteral nutrition (PN) or PN plus bovine colostrum (BC, 16–64 ml/kg/d enterally) for 5 d. In experiment 2, preterm pigs were fed PN+BC or PN+formula for 5 d. In experiment 3, preterm pigs were fed BC, formula, or human milk (HM) for 10 d. Incubator home cage activity (HCA) was quantified by continuous camera recordings.Results:Preterm birth was associated with reduced intestinal weight and HCA (experiment 1), and BC or formula supplementation increased intestinal weights and HCA (experiments 1+2). Enteral BC and HM feeding increased HCA, intestinal weights, and necrotizing enteritis resistance, relative to formula (experiment 3).Conclusion:Preterm pigs show decreased physical activity, and the first enteral feeds diet dependently stimulate both gut growth and physical activity. The effects may arise from maturation of digestive, metabolic, and neurological functions, including gut serotonin production, by the first enteral feeds and milk bioactive factors.

Reduced ex vivo stimulated IL-6 response in infants randomized to fish oil from 9 to 18 months, especially among PPARG2 and COX2 wild types

We investigated whether n-3 LCPUFA affected immune function in late infancy and explored effect-modification by single nucleotide polymorphisms (SNPs) and links to intestinal microbiota. Infants (n=105) were randomized to fish oil (FO, 1.2g/d n-3 LCPUFA) or sunflower oil (SO)-supplements from age 9-18 months. Immune function was assessed by ex vivo cytokine production in stimulated blood and plasma immunoglobulin E (IgE). We genotyped functional SNPs in PPARG2 and COX2 and analyzed fecal microbiota by 16S-rRNA terminal restriction fragment length polymorphism. FO compared to SO reduced Lactobacillus paracasei-stimulated IL-6 at 18 months (P=0.03, n=104). This effect was most pronounced among infants wild-type for PPARG2-Pro12Ala and/or COX2-T8473C (P<0.05). Predominant bacterial fragments were associated with 18 months IgE in all infants (P=0.004) (bp100) and with IL-6 production among infants weaned before 9 months (P=0.047) (bp102). Thus, FO reduced IL-6 in a genotype-modified manner. The microbiota was partly linked to IL-6 and IgE, not directly to FO.

Bioactive Whey Protein Concentrate and Lactose Stimulate Gut Function in Formula-fed Preterm Pigs

Objective: Formula feeding is associated with compromised intestinal health in preterm neonates compared with maternal milk, but the mechanisms behind this are unclear. We hypothesized that the use of maltodextrin and whey protein concentrates (WPCs) with reduced bioactivity owing to thermal processing are important factors. Method: Ninety-two cesarean-delivered preterm pigs were fed increasing doses of formulas for 5 days (24–120 mL · kg−1 · day−1). In experiment 1, 4 groups of pigs (n = 15–16) were fed lactose- or maltodextrin-dominant formulas (lactose/maltodextrin ratios 3:1 or 1:3, respectively), containing WPC with either high or low levels of IgG (WPC1 or WPC2, respectively). In experiment 2, 2 groups of pigs (n = 15–16) were fed lactose-dominant formulas with either a bioactive WPC (BioWPC, produced by reduced thermal-processing) or a conventional WPC (ConWPC). Results: In experiment 1, pigs fed formula with WPC1 had higher villi, hexose absorption, and lactase activity in small intestine, relative to WPC2, but predominantly with the lactose-dominant formula (all P < 0.05). In experiment 2, the BioWPC product had higher bioactivity, as indicated by higher IgG, lactoferrin, and TGF-&bgr;2 levels, and better enterocyte proliferation in vitro. Pigs fed the BioWPC formula showed better feeding tolerance and higher intestinal villi and lactase activity (all P < 0.05). The BioWPC formula-fed pigs also had greater physical activity (P < 0.05 on day 4) and tended to show improved hexose absorption and decreased gut permeability (both P ⩽ 0.09). Conclusions: Infant formulas containing lactose as the main carbohydrate, and WPC with reduced thermal processing, may support gut maturation and health in sensitive, preterm neonates.

Physical Activity and Gastric Residuals as Biomarkers for Region-Specific NEC Lesions in Preterm Neonates

Background: Necrotizing enterocolitis (NEC) is a serious feeding-related inflammatory gut disease with high mortality. Early clinical markers of NEC are of great importance for optimizing preventive interventions. Objective: Using preterm pigs as models, we hypothesized that an early postnatal onset of NEC can be predicted by decreased physical activity during the first few days after birth. Methods: Cesarean-delivered preterm pigs were fed parenteral nutrition and increasing amounts of formula for 5 days after birth (n = 120). Their physical activity was quantified by a continuous camera surveillance system and they were evaluated twice daily for clinical signs of apathy, discoloration, respiratory distress, abdominal distension and diarrhea. The volume of gastric residuals and the presence of macroscopic NEC-like lesions in the stomach, intestine and colon were recorded at euthanasia on day 5. Results: Half of the pigs (48%) showed clear NEC-like lesions on day 5, and these individuals had more adverse clinical symptoms from day 3 but decreased physical activity already from day 2 relative to the unaffected pigs (both p < 0.05). Only animals with NEC lesions in the small intestine had lower physical activity on days 2 and 3, and the increased volume of gastric residuals was specifically related to colon lesions (both p < 0.05). Conclusions: Decreased physical activity precedes the clinical symptoms of NEC in the small intestine of preterm pigs, and increased gastric residuals predict NEC lesions in the colon. Physical activity and gastric residuals may function as clinical biomarkers for region-specific NEC lesions in preterm neonates.

Fish oil supplementation from 9 to 18 months of age affects the insulin-like growth factor axis in a sex-specific manner in Danish infants

Several studies have investigated the effects of fish oil (FO) on infant growth, but little is known about the effects of FO and sex on insulin-like growth factor-1 (IGF-1), the main regulator of growth in childhood. We explored whether FO v. sunflower oil (SO) supplementation from 9 to 18 months of age affected IGF-1 and its binding protein-3 (IGFBP-3) and whether the potential effects were sex specific. Danish infants (n 115) were randomly allocated to 5 ml/d FO (1·2 g/d n-3 long-chain PUFA (n-3 LCPUFA)) or SO. We measured growth, IGF-1, IGFBP-3 and erythrocyte EPA, a biomarker of n-3 LCPUFA intake and status, at 9 and 18 months. Erythrocyte EPA increased strongly with FO compared with SO (P<0·001). There were no effects of FO compared with SO on IGF-1 in the total population, but a sex × group interaction (P=0·02). Baseline-adjusted IGF-1 at 18 months was 11·1 µg/l (95% CI 0·4, 21·8; P=0·04) higher after FO compared with SO supplementation among boys only. The sex × group interaction was borderline significant in the model of IGFBP-3 (P=0·09), with lower IGFBP-3 with FO compared with SO among girls only (P=0·03). The results were supported by sex-specific dose-response associations between changes in erythrocyte EPA and changes in IGF-1 and IGFBP-3 (both P<0·03). Moreover, IGF-1 was sex specifically associated with BMI and length. In conclusion, FO compared with SO resulted in higher IGF-1 among boys and lower IGFBP-3 among girls. The potential long-term implications for growth and body composition should be investigated further.