Mother's own milk (MOM) fortified with a multicomponent human milk fortifier (HMF) is recommended for preterm infants born <1500 g to achieve satisfactory weight gain and neurodevelopment (1). Although preterm formula offers the advantage of faster short-term growth, it is associated with an increased risk of necrotizing enterocolitis and late-onset sepsis (2,3) and so is generally used only in the absence of human milk.
Gastric emptying, feeding tolerance, and the attainment of full enteral feeding are enhanced by human milk (1,4–6), although the mechanisms for these are poorly understood, primarily because of a limited understanding of how milk composition influences preterm gastrointestinal motility. Enterogastric neural reflexes and nutrient and osmotic receptors trigger an inhibitory feedback system that regulates the rate of gastric emptying to ensure adequate time for digestion and absorption of macronutrients (7,8). Knowledge of the effects of differing feed compositions on the release of enterogastrones and subsequent gastric emptying is limited in the preterm population (9–11).
Breast milk composition varies widely both within and between women, particularly with regard to fat and protein (12,13). Although breast milk is the recommended source of infant nutrition, there are no published studies examining the effect of different breast milk biochemical compositions on gastric emptying in preterm infants. The variability of breast milk with respect to its biochemical and caloric content suggests that gastric emptying may vary both between and within infants according to the composition of individual feeds, particularly after fortification.
Fortification of milk for preterm infants is virtually universal; however, evidence for the effect of HMF on gastric emptying is limited and inconclusive (14–17). With limited published evidence and the wide variations in breast milk and HMF composition, it is largely unclear whether fortification affects gastric emptying in the preterm infant.
Knowledge of the composition of each feed is crucial to enable exploration of the biochemical factors that may contribute to intraindividual and interindividual differences in gastric emptying between feeds of unfortified and fortified MOM. Therefore, in the present study, we aimed to do the following:
- Investigate whether specific biochemical compositions and energy concentrations influence gastric emptying of unfortified and fortified MOM
- Determine whether gastric emptying differs between feeds of unfortified MOM and feeds of MOM fortified with S-26 HMF or FM 85 HMF, when infants are fed the same volume under similar conditions
- Explore infant and feed characteristics that influence gastric emptying of unfortified and fortified MOM
Medically stable infants between 28 and 34 weeks gestation receiving full enteral feeds of MOM were recruited from the Special Care Nurseries of King Edward Memorial Hospital, Western Australia. Infants with congenital abnormalities, gastrointestinal disease, or symptoms of feeding intolerance within the previous 24 hours were excluded. For multiple births, only 1 sibling was recruited to prevent confounding of results.
The ethics committees of the Women and Newborn Health Service and the University of Western Australia gave approval for the study. Parents were provided with verbal and written study information and provided signed informed consent.
Paired unfortified and fortified MOM feeds were studied for each infant, with the order of fortification status randomized and interfeed interval <72 hours. Attending clinicians prescribed the feed volume, frequency, and positioning of each infant. The same infant position (supine, right lateral, or prone) was maintained for each of the paired feeds during feed delivery and postprandially. Study feeds coincided with scheduled feed times.
Ultrasound scans were performed immediately postfeed delivery (T0), at 30 minutes after completion of feed delivery (T30), and at 30-minute intervals thereafter until the next feed was due, that is, T90 for 2-hourly feeds and T150 for 3-hourly feeds. Sterile scan gel (PDI, New York, NY) was prewarmed in the infants’ incubators, or used at room temperature for infants in cots.
Intragastric tube feeds were delivered by gravity, with prefeed gastric aspirates tested for acidity to confirm correct tube placement. Feeds were given at room temperature. All of the infants maintained their body temperature within normal limits and tolerated the study procedure well.
Two HMFs were used during the course of the study because of a change in practice in the study setting. S-26 HMF was added at the recommended dose of 1 sachet per 50 mL breast milk (Wyeth Nutrition, Baulkham Hills, NSW, Australia), and FM 85 HMF was added at the recommended dose of 5 g per 100 mL breast milk (Nestlé Nutrition, Rhodes, NSW, Australia). The protein content of the 2 HMFs differed; S-26 HMF contained partially hydrolyzed casein and whey proteins at a reported ratio of 40:60, whereas FM 85 contained 100% extensively hydrolyzed whey protein.
Analysis of Feed Composition
Unfortified MOM samples (3–5 mL) were collected immediately before the addition of HMF on the morning of the study. Samples were stored in sterile polypropylene capped tubes (Sarstedt, Nümbrecht, Germany) at 4°C until delivery of the study feed, and then frozen at −20°C for later biochemical analysis. One milk sample was collected when consecutive unfortified and fortified MOM feeds were prepared from the same pool of MOM. When studies of fortified and unfortified MOM occurred on separate days, milk samples were collected on each day.
Milk samples were analyzed to determine total protein (g/L), casein, whey, lactose, and fat, and the results were used to calculate energy concentration as calories per 30 mL (cal/30 mL) using standard laboratory assay methods as described by Khan et al (18). Biochemical analysis of MOM feeds containing HMF was not possible because our laboratory assays have not been validated for partially and extensively hydrolyzed proteins, and manufacturers do not disclose commercial HMF composition details. The absence of information regarding the specific mixture of amino acids and peptide bonds in HMF precludes the development of suitable protein assays for fortified breast milk. Therefore, the total biochemical and energy concentrations of fortified feeds were calculated by adding the composition data provided by the HMF manufacturers to the measured unfortified MOM concentrations.
Ultrasound Examination and Calculation of Stomach Volume
Real-time 2-dimensional ultrasound was performed using portable ultrasound (Sonologic SonoScape S6, QLD, Australia) with a 5PI phased array transducer. To acquire images for the calculation of stomach volume, the transducer was positioned perpendicular to the left lateral chest wall and rotated so that the stomach was at both its maximum diameter and length (longitudinal plane), and a clear view of the spleen was achieved. A transverse image was acquired perpendicular to the longitudinal plane. Scanning duration was <3 minutes at each time point. Images were recorded on the ultrasound machine and transferred to a laptop for measurement.
Image measurements of the longitudinal, transverse, and anteroposterior axes were made using “Screen Calipers,” version 4.0 (Iconico Inc, New York, NY) by the first author, who was blinded to details other than the infants’ study identification numbers and study dates. Stomach volume was calculated using the following equation, which has been assessed to have adequate repeatability and reliability (19,20):
Spheroid : longitudinal axis × anteroposterior axis × transverse axis × 0.52
The initial study design required recruitment of 30 infants to receive paired feeds (1 fortified MOM, 1 unfortified MOM) under identical conditions within paired feeds. A change in nutrition policy resulted in discontinuation of S-26 HMF when 12 infants had been monitored for paired fortified and unfortified feeds. Recruitment ceased during the transition to FM 85 HMF, and recommenced with the aim of recruiting a similar number of feed pairs in which the new fortifier had been used. Interim analysis of a previous preterm gastric emptying study indicated that 14 paired feeds provide an adequate sample to detect significant differences in gastric volume measurements, and so recruitment ceased when 14 unfortified/FM 85 HMF fortified pairs had been studied (21).
All of the analyses were performed using R 2.15.2 for Mac OS X (22). Additional packages nlme (23) and lattice (24) were used for linear mixed modeling and graphical exploration of the data, respectively. Descriptive statistics are presented as median (range) for infant and feed characteristics, and as mean and standard deviation (SD) for all of the other measures, unless otherwise specified. We considered P < 0.05 to be significant, and values <0.001 reported as P < 0.001. Predictors were retained in multivariate models only if they were significant at the P < 0.05 level, and borderline results (0.05 < P < 0.1) are discussed.
Both measured stomach volumes and proportions of delivered feed volume (% feed volume) were analyzed to provide both specific individual measures of emptying during feeding and comparisons between feeds of different volumes. The effect of fortification on stomach volumes and % feed volumes were examined using linear mixed-effects modeling with milk fortification status and time as predictors, and individual infants as the grouping variable. Variables for the initial model were determined by identifying all of the predictors that were significant after accounting for time and fortification status. Differences in prefortification composition between the 10 feeds that were not studied sequentially were accounted for in the multivariable model.
To test for nonlinear effects of postfeed time, 2 formulations were considered: time as a 2° polynomial and time as a categorical variable. Because the 2-hourly feeds did not have the same number of measurements as the 3-hourly feeds, the quadratic model was chosen. This was retained as the default in all of the models, testing the curve and trend separately.
Starting from a model in which all of the univariately significant predictors were included, the final model was selected by sequentially omitting nonsignificant variables until all of the remaining fixed-effects variables had marginal P < 0.05, and then testing for significance of each of the omitted variables when added to the present model. If additional significant variables were found, this process was repeated. In the situation in which 2 covariates were significant when added to the present model individually, but neither were significant when both were included, both were retained in the model to account for their effects.
Considered covariates for influences on measured stomach volumes and calculated proportions of feed delivered were birth gestation, birth weight, appropriateness of weight for gestational age, sex, postnatal age, corrected gestational age, infant weight, feed volume, feed frequency, feed milliliters per kilogram, pacifier use during feeding, infant body position, use of continuous positive airway pressure (CPAP), feed energy value (cal/30 mL), and the following biochemical concentrations measured as total grams per liter: protein, casein, whey, carbohydrate, lactose, and fat. Appropriateness of the models was assessed with a visual inspection of residual plots.
Differences with respect to fortification status in frequencies of empty stomachs, residuals >3 mL, and residuals >30% of delivered feed volume were tested for using the Fisher exact test; these were performed separately for the 2 feed frequencies. Influences on residual stomach volumes were assessed using linear mixed-effects modeling with milk fortification status, feed volume, and feed duration as predictors and individual infants as the grouping variable.
Of the 26 infants recruited, 1 infant received 1 feed of S-26 fortified pasteurized donor human milk because of insufficient MOM and so was excluded from the analysis, leaving data for 25 pairs of fortified and unfortified MOM feeds (S-26 HMF: n = 11; FM 85 HMF: n = 14). Milk composition data were not available for 3 feeds (1 unfortified, 2 S-26 fortified) from 2 infants, leaving 23 pairs of feeds for analysis. Missing data occurred when infants required care at the time of the scheduled scan (T120, n = 1; T150, n = 5).
For infants (14 girls, 11 boys) included in the analysis, the following values (mean ± SD [range]) were obtained: birth gestation 30.1 ± 1.4 weeks (28–32.9), birth weight 1331 ± 275 g (910–1910), corrected gestational age 33.1 ± 1.3 weeks (31.4–36), and postnatal age 21 ± 9 days (10–42). Six infants were small for gestational age (birth weight <10th centile), with the remainder appropriately grown. Age at commencement of full enteral feeds was 10 ± 4 days (6–20), delivered feed volume 27 ± 9.4 mL (14–42), feed mL/kg 16 ± 3.2 (12–22), and feed duration 13 ± 4 minutes (7–20). Infants receiving 2-hourly feeds were younger and smaller than those receiving 3-hourly feeds (Table 1). Two infants received nasal CPAP. Prescribed medications including caffeine, vitamin D, ferrous sulfate, nystatin, and probiotics were administered to study infants at the prescribed times: none of these are known to affect gastric emptying. Infants did not receive the same medications at each of the paired study feeds. Because the combined volume of administered medications was typically ≤1 mL, it is unlikely that this would influence differences in gastric volume measurements.
Total biochemical and energy concentrations are reported in Table 2, with significant differences observed between fortified and unfortified MOM for mean carbohydrate, protein, whey, casein, and energy concentrations.
Influences on Gastric Emptying During Feeding
Immediately postfeed, the retained feed proportion differed by the total casein concentration of the feed and by milk fortification status. For each additional 1 g/L of casein, the retained feed proportion was 2.6% lower (P = 0.007). Compared with unfortified MOM, FM 85 fortified feeds had retained feed proportions on average 11% higher (P = 0.006) and stomach volumes 2.8 mL higher (P = 0.002), whereas S-26 fortified feeds had stomach volumes 3.6 mL lower (P = 0.014; Table 3).
The T0 stomach volume was consistently measured less than the delivered feed volume. For an average feed volume of 30 mL, the average T0 volume was 21 mL. Larger feed volumes were associated with larger T0 stomach volumes, with each additional 1 mL of delivered feed volume resulting in an additional 1.8 mL at T0 (P < 0.001).
T0 stomach volumes and retained feed proportions were influenced with feed frequency and postnatal age, feed mL/kg, and CPAP. Compared with 3-hourly feeds, the retained feed proportion was on average 13.4% higher for 2-hourly feeds (P < 0.001). After accounting for feed volume, small but significant effects of postnatal age and feed mL/kg were observed. Stomach volumes were 1.3 mL lower for each additional postnatal week (P = 0.031), with a trend toward lower percentage feed volumes (P = 0.065), and were 1 mL lower for each 1 mL increase in feed mL/kg (P = 0.03). For the 2 infants receiving CPAP, retained feed proportions were 36% higher (P < 0.001) than those not receiving respiratory support.
Influences on Postprandial Gastric Emptying
S-26 fortified feeds emptied similarly (P = 0.23) to unfortified MOM, whereas retained feed proportions of FM 85 fortified feeds were on average 5.6% higher (P < 0.001) across the postprandial period. Furthermore, infants fed FM 85 fortified MOM every 2 hours had retained feed proportions on average 8.3% higher than those fed every 3 hours (P = 0.002). Although feeds of unfortified MOM and of S-26 fortified MOM had curvilinear patterns of gastric emptying, with the rate of emptying slowing over time, feeds of FM 85 fortified MOM emptied at a more consistent rate that approximated a linear pattern of emptying (P = 0.046; Fig. 1). Stomach volumes decreased over time, with the emptying rate decreasing as the stomach emptied (P < 0.001).
Infant positioning and feed frequency had large influences on gastric emptying throughout the postprandial period, whereas infant weight and feed volume had small but significant effects. Infants positioned supine had retained feed proportions on average 11% higher than those in prone or right lateral positions (P < 0.001), whereas infants fed every 2 hours had retained feed proportions on average 10.4% higher than those fed every 3 hours (P = 0.012). For each 100 g increase in infant weight, the retained feed proportion was 2.5% higher (P < 0.001), and larger feed volumes were associated with higher retained feed proportions over time (P < 0.001). There was a nonsignificant trend toward faster postprandial emptying in small for gestational age infants, with retained feed proportions on average 6% lower across the postprandial period (P = 0.053).
In constructing the multivariable model for postprandial gastric emptying, individually there were significant effects of infant weight and feed mL/kg, but on including both in the model, 1 cancelled out the effect of the other, suggesting a relation between the variables. This is explained by the use of infant weight in the calculation of feed mL/kg.
Gastric Residual Volumes
Empty stomachs were less frequent for 2-hourly feeds (29% at T90, n = 24) than for 3-hourly feeds (62% at T150, n = 21; Table 4). For 3-hourly feeds, empty stomachs were more prevalent for unfortified feeds (83%, n = 12) than for fortified feeds (FM 85 HMF: 43%, n = 7; S-26 HMF: 0%, n = 2); assuming that missing data for fortified feeds were for empty stomachs, the incidence of empty stomachs, 56% (n = 9) and 50% (n = 4), respectively, would be considerably lower than for unfortified MOM.
Higher residual volumes were associated with larger feed volumes (P = 0.043) and shorter feed delivery (P = 0.058) for 2-hourly feeds but not for 3-hourly feeds. After accounting for these in 2-hourly feeds, S-26 fortified feed residuals were similar to those of unfortified feeds (P = 0.179), and FM 85 fortified feed residuals were significantly higher (average 2 mL higher, P = 0.015; Table 4). Similarly, FM 85 fortified feed residuals were significantly higher (average 1.1 mL higher, P = 0.040) for 3-hourly feeds. Residual stomach volumes >3 mL were observed in 46% of 2-hourly feeds and were infrequent following 3-hourly feeds (Table 4).
One gastric residual volume >30% feed volume was observed following a fortified 2-hourly feed (Table 4). Accounting for feed duration, the effect of fortifier on the retained % feed volume was significant, with the retained % feed volume for FM 85 fortified feeds on average 9.9% higher than for unfortified feeds of the same duration (P = 0.018), although the effect of feed duration was not significant (P = 0.076), and the effect was small (∼1% for each additional minute of feed duration). Feed volume was not associated with gastric residuals as % feed volume remaining in the stomach.
The present study has shown that the biochemical composition of breast milk feeds influences gastric emptying in preterm infants, with higher MOM casein concentrations associated with faster gastric emptying during delivery of intragastric tube feeds. FM 85 fortified feeds emptied more slowly than unfortified feeds both during feed delivery and across the postprandial period, whereas S-26 fortified feeds emptied faster during feed delivery (Table 3). The differences are likely explained by the stimulation of the ileal brake, which alters according to the dose and chemical structure of the various end products of digestion (8). Although statistically significant, the magnitude of the effects of MOM casein concentration and HMFs on gastric emptying is small and not of clinical concern for the stable preterm infant.
Faster emptying of MOM feeds of higher casein concentrations during feed delivery may be explained by the more extensive intragastric hydrolysis of caseins than of whey proteins, which results in rapid emptying of high casein feeds during feed delivery without stimulating enterogastrone release (25). Subsequent slower transit is likely explained by triggering of the ileal brake in response to detection of the increasing postprandial nutrient load and/or other triggers. Because faster emptying was not sustained, it is unlikely to influence feeding tolerance in the preterm infant.
Feeds of S-26 fortified MOM, with a whey:casein ratio of 60:40, had lower postfeed stomach volumes than unfortified feeds, although retained feed proportions were similar, suggesting that the association is not clinically significant. Typically, gastric emptying of fortified MOM is slower or similar to unfortified milk (14–17).
Human milk caseins are more extensively hydrolyzed in the stomach than bovine caseins because of differences in physicochemical structures (26–28). The structure of hydrolyzed proteins in HMF is not known; however, partial hydrolysis of HMF bovine caseins could result in structures comparable to human milk caseins, resulting in similar gastric emptying rates and feeding tolerance of unfortified and S-26 fortified MOM feeds.
The gastric transit of FM 85 fortified feeds was slower than that of unfortified feeds both during feed delivery and postprandially. This agrees with the literature (14), in which the FM 85 fortified milk had a significantly slower gastric half-emptying time (48 minutes vs 21 minutes) and a more linear pattern than unfortified milk. In spite of finding statistically higher retained feed proportions at each time point, no complications were observed in our stable preterm infants, although it is not possible to predict the impact on feeding intolerance in susceptible infants (29). Slower emptying of FM 85 fortified feeds may be because of the HMF protein content that consists solely of extensively hydrolyzed whey protein, which increases glucose-dependent insulinotropic polypeptide secretion (27,30). In turn, glucose-dependent insulinotropic polypeptide is associated with increased gastric secretions and delayed gastric emptying, thus explaining in part our results.
Feed osmolality may also have played a role in the slower gastric transit of FM 85 fortified MOM (31). Hyperosmolar feeds stimulate duodenal osmoreceptors that inhibit gastric emptying and increase gastric secretions (7). Although measurement of osmolarity was outside the scope of the present study, the osmolar range for unfortified preterm breast milk is 263 to 370 mOsmol/L (17,32). FM 85 fortified preterm breast milk has a reported osmolarity of 356 mOsmol/L (Nestlé Nutrition); however, clinical studies report values as high as 472 mOsm/L (95% confidence interval 428–545 mOsm/L) (31). Because the recommended maximum osmolarity for infant feeds is 400 mOsmol/L (7), it is possible that the addition of HMF caused a hyperosmolar load, resulting in slowed gastric emptying.
Feed energy concentrations did not influence gastric emptying most likely because of the relatively small differences between unfortified and fortified feeds, so the ileal brake was not stimulated. Furthermore, the evidence is conflicting as to whether energy concentrations do affect gastric emptying in preterm infants, with 1 study reporting no differences between feeds in the range of 5 to 20 cal/30 mL, and another reporting higher retained feed proportions for feeds of 20 and 24 cal/30 mL at 80 minutes postfeed (33,34).
Both feed volume and infant positioning influenced gastric emptying, although the observations are based on interindividual differences. Larger 3-hourly feeds emptied more quickly than smaller 2-hourly feeds. This is consistent with the evidence from adult and pediatric studies that higher feed volumes empty more rapidly (35,36), with scant data available for preterm infants (34). Accounting for feed volume, infants positioned prone had faster gastric emptying than those positioned supine. Evidence for the effect of positioning is inconsistent, with 2 of 4 published studies reporting no intraindividual difference in gastric emptying between supine and prone positions (37–40), suggesting the need for more robust investigations.
Increasing postnatal age and feed milliliters per kilogram were associated with faster emptying during feed delivery, whereas increasing infant weight was associated with slower postprandial emptying, although the magnitudes of the effects were small.
Final residual volumes are routinely measured via aspiration, and volumes >3 mL together with other markers may prompt closer monitoring for feeding intolerance (41). Mean gastric residual volumes in the present study were typically <2.5 mL, as expected for the stable preterm infant. Although FM 85 fortified feeds were associated with higher residual volumes, the actual volume difference was small and therefore not considered problematic (Table 4). Empty stomachs were more common for 3-hourly feeds (2-hourly feeds: 29%; 3-hourly feeds: 62%) and for unfortified MOM (2-hourly feeds: 33%; 3-hourly feeds: 83%); it is likely that one third to two thirds of infants may have an empty stomach and cue for a feed before the scheduled feed time. In these cases, consideration should be given to flexible feeding times because cue-based feeding is associated with a more rapid transition to full oral feeding (42).
In the absence of a fluid prefeed gastric aspirate, it is usually assumed that the healthy tube-fed preterm infant has an empty stomach before feeding; however, we have shown with ultrasound that residual curd is often present (21). It is possible that the presence of stomach contents is advantageous for infants, considering the fact that the healthy human fetus’ stomach is never empty, and early enteral feeding instead of fasting results in a better feeding tolerance (43,44). The presence of a small gastric residual enables continuous gastric emptying of breast milk feeds, which may contribute to the development of gastrointestinal function and the intestinal microbiome.
Higher casein concentrations in MOM feeds are associated with faster gastric emptying during feed delivery, but not in the subsequent postprandial period. The biochemical composition of HMF influences gastric emptying in which postprandial emptying of S-26 fortified MOM is similar to that of unfortified MOM, whereas FM 85 fortified MOM empties more slowly both during feeding and in the postprandial period. The size of these effects, although statistically significant, is not of clinical concern, making it unlikely that either MOM milk composition or fortification with S-26 or FM 85 HMF contributes to feeding intolerance in the stable preterm infant.
Of the Hickets
Londoner Nicholas Culpeper (1616–1654) wrote a 39-page addenda to his Directory for Midwives called A Tractate of the Cure of Infants, in which he addresses the problem of hickets [hiccups]:
It comes from corruption of the food in the stomach or from milk filling it or from cold air; these hurt the expulsive faculty and it is stirred up to expel what is harmful…. Hickets is commonly not dangerous in children and cease when the cause is taken away. If it can be from a vehement cause and goes to the nerves there follows a Convulsion or Epilepsie and death. That from corruption of nourishment is cured by vomit with a feather dipt in Oyl to tickle the throat; then strengthen the Stomach with hot things.
Nicholas Culpeper, student in physics and astrology. Courtesy Wikimedia Commons.
—Contributed by Angel R. Colón, MD
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