Main

The first step of fat digestion occurs in the stomach and is catalyzed by lingual or gastric lipase. These enzymes are similar in structure and characteristics, but differ in origin among species which is either oral(pregastric esterase in ruminants and lingual lipase mainly in rodents), or gastric (rabbit, dog, guinea pig, human)(1, 2). These lipases are well developed at birth(1–4) and may be of greater importance in the newborn for two reasons: the sudden change from the high carbohydrate diet of the fetus to the high fat diet of the newborn and the immaturity of exocrine pancreatic function(1, 2, 5, 6). Furthermore, initial action by gastric lipase is essential for the subsequent digestion of the triglyceride in human milk fat globules(7–9) or formula fat emulsions(10) by pancreatic lipase or milk bile salt-dependent lipase, an enzyme(8, 9) that contributes to fat digestion in the newborn.

During the neonatal period there is high demand for an adequate energy supply as well as for essential nutrients. Fat is essential for the newborn and especially the premature infant for several reasons: it provides more energy than other nutrients, it can be stored in the body in considerably larger amounts than carbohydrates and proteins, it provides essential fatty acids, and it is the only vehicle for fat-soluble vitamins(11).

Fat is supplied mainly from mother's milk or formulas which contain 45-50% of total calories as fat. The fatty acid blend of the former is most appropriate for the fatty acid requirements of preterm infants(11). Commercial formulas(12, 13) contain either mainly LCT, or up to 50% MCT. The fatty acid composition of some LCT formulas is more similar to human milk, and thus probably more appropriate to the premature infant's needs(14). The advantage of MCT in formulas is thought to be associated with their more efficient digestion (medium chain fatty acids are readily released from glycerides by gastric and pancreatic lipases), absorption (less dependent on micellar solubilization by bile salts), and transport (principally through the portal vein to the liver for oxidation rather than through the intestinal lymphatics)(15).

The objective of this study was to evaluate gastric function (lipase and pepsin activities, pH, and volume) as a function of the diet of premature infants, and to assess whether the activity of gastric lipase and fat digestion in the stomach are modulated by the nature of the diet (human milk, LCT formula, or MCT formula).

METHODS

Subjects and diets. Twenty-eight infants (12 girls and 16 boys) were enrolled in the study after informed consent was given by the parents. The experimental protocol was approved by the Institutional Review Board of Georgetown University Hospital. All infants were appropriate for gestational age (24-34 wk)(16), had a birth weight in the range of 0.5 to 1.7 kg, and were 1-11 wk of age at the time of the study (average 5-6 wk) (Table 1). The infants were fed either their own mother's milk (n = 11) or one of two randomly selected formulas,i.e. SMA Super Preemie formula (SMA SP) (Wyeth Ayerst Laboratory, Philadelphia, PA) (n = 9) or Similac Special Care formula (Similac SC) (Ross Laboratory, Columbus, OH) (n = 8). The study was initiated when the infants had received their diet for at least 1 wk as bolus feedings by naso- or orogastric tube (eight feeds/d, one feeding each 3 h). The infants were studied one to five times at weekly intervals and, when studied several times, an average of the data was used to represent each infant. There was no significant difference in the mean postconceptional ages (= gestational age plus age at the time of the study) of the infants studied once (34.9 ± 1.9) and those studied several times (35.4 ± 2.6).

Table 1 Clinical data of infants
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The nutrient composition of the three diets was comparable(Table 2). Human milk contains slightly lower levels of protein and carbohydrate than do the formulas. The SMA SP formula is a LCT formula with a fatty acid blend (20% oleo, 25% safflower, 27% coconut, 18% soy oils, and 10% MCT) similar to that of human milk except for the absence of docosahexaenoic (22:6n - 3) and arachidonic (20:4n - 6) acids (Table 3). The amount of long chain mono- and polyunsaturated fatty acids represents about 50% of the total fatty acids in either human milk or SMA SP. The saturated medium chain (C8:0-C12:0) fatty acids are higher in SMA SP-formula than in human milk, 27 versus 12%. The Similac Special Care formula has a fat blend of 50% MCT, 30% soy oil, and 20% coconut oil, providing about 55% medium chain fatty acids and 22% polyunsaturated fatty acids. The polyunsaturated: saturated fatty acid ratio was close for all diets, ranging from 0.31 to 0.33. In the formulas, carbohydrates are composed of equal amounts of lactose and glucose polymers, and the proteins consist of a mixture of whey proteins and casein.

Table 2 Composition of human milk and infants formulas
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Table 3 Fatty acid composition of human milk and formulas
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Feeding volumes were similar among the three groups: per meal 18.4 ± 0.5 mL of human milk, 17.4 ± 0.5 mL of SMA SP, and 17.0 ± 0.7 mL of Similac SC per kg of body weight. All feeds had similar pH, 6.6 ± 0.1, 6.4 ± 0.5, and 6.5 ± 0.1 for human milk, SMA SP, and Similac SC, respectively.

During the entire study period, the infants received 126 ± 5, 114± 4, and 111 ± 6 kcal/kg/d, and daily fat intakes were 5.9± 0.2, 6.0 ± 0.2, and 5.5 ± 0.2 g of fat/kg for human milk, SMA SP, and Similac SC, respectively. Volume of feeds, energy, and fat intake expressed per kg of body weight were constant as a function of postnatal age (from the 1st wk to the 5th wk of bolus enteral feeding). None of the infants received medication orally with study feedings.

Collection of gastric contents. The study was initiated when the infants received bolus feedings by naso- or orogastric tube. Studies were conducted in the morning (0800-12 noon) and were started 3 h after the end of the previous meal. The tube (French #8) was passed to the stomach and secured. The feedings were given over 5-7 min (6.9 ± 1.0, human milk, 5.2± 0.5, SMA SP, and 5.1 ± 1.5, Similac SC). Samples were taken 10, 30, and 50 min after feeding started, timed from the time when half of the volume was fed. At each time point, the entire stomach contents were aspirated into a syringe, and the volume was measured at a precision of 0.5 mL. A 2-mL sample was taken for pH measurement (portable pH meter, Sper Scientific#840008, CMS, Houston TX) and subsequent analyses, and the remainder was immediately refed to the infant. A mixture of inhibitors(17) (10% vol/vol) was added to the sample aliquot taken for neutral lipid analysis in order to prevent lipolysis during storage. Under these conditions, 85-90% of gastric lipase activity and 95-96% of BSDL activity were inhibited (data not shown).

To obtain a gastric fasting sample (prefeed sample) and not to affect the gastric content of lipase on the days when fat digestion was studied, the fasting sample was obtained about 3 h after and immediately preceding the following feed on the morning before or after the experiment. The pH, volume, and appearance were recorded, and the pH of the sample was adjusted when necessary to pH 4.0-4.5 with a few drops of 0.1 N NaOH to prevent the inactivation of gastric lipase(18). This pH range does not affect pepsin activity, which is stable when stored at pH 1-5. All specimens were rapidly frozen (dry ice) and kept at -70 °C until analysis.

Quantification of lipase activity. Gastric lipase activity in the aspirates was quantified using as substrate a stable emulsion of tri[3H]olein(3, 4). Ten to 30 μL of gastric aspirate diluted 10-fold (three aliquots of different volume in order for at least two determinations within the linear range) were incubated in an assay system containing 1 μmol of labeled triglyceride, 10 μmol of sodium acetate-acetic acid buffer (pH 5.4), and 7 mg of bovine plasma albumin(Sigma, Chemical Company, St. Louis, MO) in a final volume of 200 μL. Incubation was in a Dubnoff shaking bath for 30 min at 37 °C. The[3H]oleic acid produced was separated by liquid-liquid partition(19) and quantified in a Beckman liquid scintillation counter (Beckman model LS-7500, Beckman Instruments, Inc., Fullerton, CA). Lipase activity is expressed as micromoles of FFA released per min(international units). Gastric lipase output (concentration × volume) is expressed as units/kg of body weight.

BSDL activity was measured in human milk and in 12 specimens of gastric contents collected after feeding human milk, as previously described(20, 21). Briefly, 50 μL (diluted 500 times) of milk or gastric specimens were incubated at pH 9.0 in a 200-μL assay system containing 12 mM/L taurocholate, 150 mM/L NaCl, 2.8% BSA, 60 mM/L Tris-HCl buffer, and a [3H]triolein emulsion (final concentration 1.6 mM/L) prepared in 10% gum arabic solution. Incubation was for 15 min at 37°C in a Dubnoff shaking bath. The [3H]oleic acid released was separated and quantified as described for the gastric lipase assay. BSDL activity in human milk specimens was in the range of 20-40 U/ml as reported previously(45, 46). In gastric content specimens tested under optimal conditions (pH 9.0 and 12 mM taurocholate) enzyme activity was in the same range (19-41 units), confirming earlier reports that BSDL is resistant to pH > 3.5(22) and is not destroyed by pepsin(22).

To assess whether BSDL may be active in the stomach in case of gastroduodenal reflux, i.e. in the presence of bile salts, or in the presence of FFA released by gastric lipase, incubations of human milk were carried out in vitro under similar conditions: pH 9.0, optimal for BSDL and pH 6.0 and 5.0 (that simulate the gastric environment) with 1 or 12 mM taurocholic acid and/or in the presence of oleic or palmitic acid at concentrations similar to those found in gastric contents (0.5 or 3 mM/L). The data show maximal activity of BSDL at pH 9.0 and 12 mM/L taurocholic acid. Under these conditions, addition of palmitic or oleic acid did not affect BSDL activity. In the presence of only 1 mMol/L taurocholate, no activity of BSDL could be detected, even with addition of FFA. At pH 5.0 in the presence of 1 or 12 mM taurocholate, no activity was detected even in the presence of FFA(data not shown).

Quantification of pepsin activity. Pepsin was measured in 20-40μL of gastric aspirate using hemoglobin as substrate, as recently reported(4). One pepsin unit has been defined as the amount of enzyme required to produce 0.1 μmol of tyrosine-containing peptides at 37°C in 10 min at pH 1.8 from a 2% hemoglobin solution. Pepsin output(concentration × volume) is expressed as units/kg of body weight.

Lipid analysis: determination of neutral lipids in human milk, formula and gastric aspirates. Lipids in gastric aspirates and in human milk or formula were homogenized and extracted in chloroform/methanol (2/1, v/v) containing 0.01% BHT(23). To ensure complete protonation of fatty acids, the organic solvent phases were partitioned with 20% (v/v) 0.15 M aqueous NaCl containing 2% glacial acetic acid (v/v, pH 3.0)(17). Neutral lipid classes (triglyceride, diglyceride, monoglyceride, FFA, and free cholesterol) were separated by two-stage, one-dimensional thin layer chromatography according to Bitman and Wood(24). Briefly, the chromatogram (TLC plates 19C, Si250-PA, Baker, Phillipsburg, NJ) was first developed in chloroform/methanol/ethanol/acetic acid (98/2/1/0.1, v/v/v/v) under saturated conditions at room temperature, and after air drying for 10 min, the plate was developed in hexane/ethyl ether/acetic acid (94/6/0.2, v/v/v). For staining, the TLC plates were dipped in a 10% copper sulfate-8% phosphoric acid (w/v) solution in a dipping tank for 3 s. The plates were drained for 2 min and heated in an oven at 130 °C for 30 min. Lipid was quantified by densitometry with a Shimadzu dual-wavelength densitometer (Shimadzu Scientific Instruments, Columbia, MD). Standard calibration curves were constructed from several TLC plates with 1-20 μg of lipid mixture. The densitometric area was fitted by computer to a linear regression model, the coefficient of determination, r, was in the range of 0.90-0.98. A standard sample(5-20 μg) was included in every TLC plate to correct for deviations from the standard calibration curve. Lipid recovery from TLC plates was in the range of 90.8 ± 3.1. Values were converted into moles using average molecular masses calculated according to the fatty acid composition of each diet. The extent of triglyceride lipolysis was calculated(25) as the percent of triglycerides disappearance from total glycerides present (triglyceride + diglyceride + monoglyceride)(%Tgt) and from the percentage of triglyceride originally present in human milk (98%) and formulas (99%). (%Tgt0),i.e. triglyceride disappearance (%) =% Tgt0 -% Tgt.

Collection and quantification of fecal fat. Stools were collected over a 72-h period, bracketed by charcoal markers as previously described(14, 26) in 10 of the 28 infants studied. Total fecal lipids were extracted and determined gravimetrically according to the procedure of Jeejeebhoy et al.(27). This technique ensures the quantitative estimation of lipids containing medium-chain fatty acids. The extent of fat absorption was calculated as the difference between the fat content of the feedings and the fat content of the stool and expressed as percent of consumed fat(14, 26).

Electron microscopy of milk and formula fat. To enable the visualization of lipolysis in particles that differ in structure and to assess structural changes of milk fat globules or formula fat particles during digestion, gastric samples collected from one premature infant fed either human milk or SMA SP were prepared for electron microscopy. Diet samples and post-feeding specimens were fixed in 2% osmium tetroxyde in 0.3 M sodium cacodylate buffer at pH 7.4. Post-feeding specimens were also fixed as described above but at pH 5.5. Fixation was performed in a cold room overnight with constant shaking. About 24 h later, samples fixed with osmium were concentrated into pellets by centrifuging in a Microfuge (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA) for 1 min at room temperature. The specimens were concentrated, dehydrated, and embedded in epon as previously described(28).

Statistical analysis. All assays were conducted in triplicate. Statistical comparisons were made by one-way analysis of variance (ANOVA) and Fisher's test using the Statview II microcomputer program (Abacus, Berkeley, CA). The significance level was set at p < 0.05 for all variables tested. Mean values are presented as mean ± SEM unless otherwise indicated.

RESULTS

Gastric volume, pH, lipase and pepsin activity, and output. Gastric volume, expressed as mL/kg of body weight, and pH were similar for 50 min after feeding among the three groups of infants irrespective of the diet (Fig. 1A). Fasting gastric pH (3.1-3.4) was comparable among the three groups of infants (Fig. 1B), as was the pH during the first 50 min after feeding, irrespective of the nature of the meal (Fig. 1B).

Figure 1
figure 1

Effect of diet, human milk (n = 11) or formulas containing long chain (SMA SP) (n = 9) or medium chain(Similac SC) (n = 8) fatty acids on gastric volume (A) and pH (B). Data are means ± SE. Statistical analysis: one-way ANOVA, Fisher test. There were no significant differences at any time points.

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Basal and postprandial concentrations and output of gastric lipase(expressed as gastric lipase units per kg of body weight), were similar in the three groups of infants, irrespective of diet (Fig. 2, A and B). There were no differences in lipase activity or output as a function of gender, race, or time from initiation of enteral bolus feeding (1-5 wk) (data not shown). There was no relationship between postconceptional age and lipase activity (R2 = 0.061, 0.031, 0.101 for human milk, SMA SP, and Similac SC-fed infants, respectively).

Figure 2
figure 2

Effect of diet, human milk, SMA SP, or Similac SC on gastric lipase concentration (A) and output (B). Data are means ± SE of 11 (human milk), 9 (SMA SP), and 8 (Similac SC) infants. Statistical analysis: one-way ANOVA, Fisher test. There were no significant differences at any time points.

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Basal and postfeeding pepsin concentrations and output were similar among the three groups of infants, irrespective whether fed human milk or either formula (Fig. 3, A and B). As described above for lipase, there were no differences in pepsin concentration or output as a function of gender or race. There was no relationship between postconceptional age and pepsin activity in human milk and SMA SP fed infants (R2 = 0.196 and 0.001, respectively). Whereas, in the Similac SC fed infants, such a relationship was suggested (R2 = 0.567).

Figure 3
figure 3

Effect of diet, human milk, SMA SP, or Similac SC on pepsin concentration (A) and output (B). Data are means± SE of 11 (human milk), 9 (SMA SP), and 8 (Similac SC) infants. Statistical analysis: one-way ANOVA, Fisher test. There were no significant differences at any time points.

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Fat digestion and absorption. The major component of milk or formula fat is triglyceride (98-99%), no monoglyceride or FFA were present, and diglyceride was present in trace amounts only (0.20% in human milk, 0.23% in SMA SP, and <0.1% in Similac SC. The amount of fat fed was similar irrespective of diet (5.9 ± 0.2, 6.0 ± 0.2, and 5.4 ± 0.2 g/kg/d, respectively, in human milk-, SMA SP-, and Similac SC-fed infants).

Triglyceride concentrations decreased and the products of lipolysis, FFA, and diglycerides, increased throughout the 50 min after feeding (Fig. 4). Monoglyceride levels remained low except for a slightly greater increase 50 min after feeding in the Similac SC-fed group than in either the human milk- or SMA SP-fed groups (Fig. 4). Gastric lipolysis (expressed as percentage decrease from initial triglyceride minus percentage triglyceride in the stomach at each sampling time) was significantly greater (p < 0.05) at 30 and 50 min in human milk-fed infants (16.8 ± 2.4-25.3 ± 1.8) than in infants fed either SMA SP (8.4 ± 1.3-13.1 ± 2.7) or Similac SC (10.4± 2.6-14.4 ± 4.4) (Fig. 5). Fat excretion was significantly higher (p < 0.01) in infants fed formula (0.7± 0.1 g/kg/d and 0.8 ± 0.2 g/kg/d, respectively, for SMA SP(n = 3) and Similac SC (n = 3), than in infants fed human milk (n = 4) (0.3 ± 0.1 g/kg/d). Fat absorption was not significantly different among human milk- or formula-fed infants, although it was higher in the former (94.3 ± 2.19%, n = 4) than in the latter (84.4 ± 4% 0.8, n = 3 and 84.7 ± 3.0%,n = 3, in SMA SP- and Similac SC-fed infants, respectively).

Figure 4
figure 4

Effect of diet on gastric lipolysis in human milk(n = 11) (A), SMA SP-fed (n = 9) (B) and Similac SC-fed (n = 8) (C) infants. TG; triglycerides, FFA; free fatty acids, DG; diglycerides,MG; monoglycerides. Data are mean ± SE. Statistical analysis: one-way ANOVA, Fisher test. Different superscript letters indicate significant differences between the three diets.

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Figure 5
figure 5

Digestion of human milk or formula fat in the stomach. Lipolysis is expressed as disappearance of triglyceride (TG% at zero time - TG% of total glycerides at each digestion time) of 11 human milk-fed, 9 SMA SP-fed, and 8 Similac SC-fed infants. Data are means ± SE of 11 (human milk), 9 (SMA SP), and 8 (Similac SC) infants. Statistical analysis: one-way ANOVA, Fisher test. Different superscript letters indicate significant differences between the three diets.

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Structure of lipid particles during gastric lipolysis. Electron microscopy of gastric specimens taken before digestion (Fig. 6, A and D) and 50 min after feeding either human milk or SMA SP shows that the products of lipolysis remain confined within the milk fat globules(Fig. 6, B and C, human milk) and formula fat particles(Fig. 6, D and E, SMA SP). When the amphipathic nature of the fatty acid lipolytic products is maintained during specimen preparation, the fatty acids can be visualized as electron opaque lamellae(28) and (Fig. 6B, at pH 7.4) or electron dense deposits (Fig. 6, E and F). In specimens prepared at low pH, lamellar structures may not be preserved because the protonated fatty acids become soluble in the remaining triglyceride of the milk lipid core (Fig. 6C, pH 5.5).

Figure 6
figure 6

Electron microscopy of milk fat droplets(A-C) and formula fat particles (D-F) before (A, D) and after 50 min of gastric digestion (B, C, E, F). Human milk. (A) Milk lipid was fixed at pH 7.4. Undigested lipid droplet(L). Bar = 1 μm (16,000×). (B) Milk lipid collected after 50 min of digestion was fixed at pH 7.4. Water spaces (arrows) are present within the core of the milk lipid droplet (L). Bar = 1μm (16,000×). (C) Milk lipid collected after 50 min of digestion was fixed at pH 5.5. Lipolytic products appear as small dense patches (arrowheads) in the core of the lipid droplet (L). Bar = 1 μm (16,000×). SMA SP formula. (D) Lipid was fixed at pH 7.4. Undigested lipid droplets (L). Bar = 0.5 μm(32,000×). (E) Lipid collected after 50 min of digestion was fixed at pH 7.4. Water spaces (arrows) are present within the core of the lipid droplet (L). Bar = 0.5 μm (32,000×). (F) Lipid collected after 50 min of digestion was fixed at pH 5.5. Lipolytic products appear as small dense patches (arrowheads) deposited throughout the lipid droplet (L). Bar = 0.5 μm(32,000×).

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DISCUSSION

The aims of this study were to investigate whether gastric function assessed as gastric emptying, pH and digestive enzyme activity, and intragastric lipolysis are modulated by the nature of feedings (human milk or formula) given to premature infants.

The gastric phase of fat digestion has been studied in newborns of several species, rat(29–31), rabbit(32), dog(33), ruminant(34), fed mother's milk. No comparative studies have been done, however, in premature infants fed different types of feedings to assess pre and postprandial levels of gastric digestive enzymes and the extent of lipolysis in the stomach. This study shows that the nature of the diet, and specifically the physical structure of lipid particles (i.e. large membrane bonded milk fat globules or smaller formula fat particles containing either predominantly LCT or as much as 50% MCT), does not affect the basal or postprandial level of gastric enzymes (Figs. 2 and 3). Gastric lipase concentration (4-15 U/ml) was in the range previously reported in adults(4, 35). Mean postprandial gastric lipase output throughout the 50-min study period (U/kg of body weight: 23.1 ± 5.1, 28.3 ± 6.6, and 22.5 ± 6.4 for human milk, SMA SP, or Similac SC, respectively) was comparable to the gastric lipase output of healthy adults fed a high fat diet (22.6 ± 3.0)(35). It is thus possible that the high gastric lipase level of premature infants is the result of adaptation to the high fat diet of human milk or formula. Such a diet-associated increase in gastric lipase was previously reported in animals(36–39) and recently also in human adults(35). In infants, as previously reported in animal studies(37, 38), the increase in gastric lipase activity is independent of the nature of dietary fat. The mean postprandial pepsin output (597 ± 77, 743 ± 97 and 639 ± 142 U/kg body weight in human milk, SMA SP or Similac SC-fed infants, respectively) was, however, lower in premature infants than in healthy adults(3352 ± 753 U/kg)(36). This is in agreement with previous reports of low pepsinogen secretion in premature infants compared to full term infants(4, 40).

Gastric lipase and pepsin secretion followed the same pattern in premature infants as previously reported in adults(41, 42). From regression analysis of the data presented, an overall relationship between lipase and pepsin outputs was found independent of the type of feeding (r = 0.82, p = 0.0001, n = 28). Comparable correlations were evident separately with the three diets(r = 0.70, p = 0.001, r = 0.91, p = 0.0001, and r = 0.85, p = 0.0001 for human milk, SMA SP and Similac SC, respectively).

Gastric pH, either at baseline or after feeding, was comparable for all diets and in the range previously reported(43, 44). This would suggest similar buffering capacity of human milk and formulas as well as similar gastric secretion as suggested by comparable gastric lipase and pepsin output (Figs. 2B and 3B). The optimal pH for gastric lipase is in the range of 4.0-6.0. Thus, the pH of gastric contents after ingestion of human milk or formula was in the range of optimal gastric lipase activity.

Siegel et al.(45) reported the relatively small contribution of secretions to gastric volume in premature infants. One, therefore, can assume that the volume found in the stomach represents almost entirely the remaining volume of feed. Our study shows that the rate of stomach emptying, judged by gastric volumes measured, was also not affected by the type of diet. The gastric volumes measured in this study are in good agreement with those reported by investigators using nonabsorbable markers(polyethylene glycol or technetium) to measure gastric emptying of different type of meals in premature infants(46–49). For instance, 30 min after feeding, 56, 57, and 52% of the initial volume of Similac SC, human milk, and SMA SP, respectively, has left the stomach. Cavell(49) reported that half of a human milk meal left the stomach in about 25 min in preterm infants given, as herein, a 22 mL/kg bolus feeding. We found a similar gastric emptying pattern for human milk and formulas, contrary to Cavell(49), who reported a slower gastric emptying rate for formula as compared to human milk. Our data are, however, consistent with more recent studies showing no difference in gastric emptying of feeds identical in composition and caloric density given at volumes of about 20 mL/kg(45, 48). The fatty acid nature of formula fat does not seem to affect gastric emptying rate(18, 47) (Fig. 1A). Our study shows that this finding is also true for human milk.

We found that gastric hydrolysis of human milk fat was 1.7 to 2.5 times higher than that of formula fat. A few of the human milk fed infants were occasionally fed one of the two formulas, SMA SP (n = 3) or Similac SC (n = 4). These infants provided the opportunity to study each subject as his/her own control to assess the effect of diet on gastric function. Comparable data were obtained in these infants. No differences were found in gastric lipase and pepsin levels, nor gastric pH and volume (data not shown); however, human milk was hydrolyzed to a greater extent (12-25-29%versus 4-12-17%, human milk versus SMA SP or 7-13-21%versus 4-6-10%, human milk versus Similac SC) than formula, thus reinforcing the findings in the three groups of infants fed either human milk or the two types of formula. The greater extent of hydrolysis of milk compared with formula fat cannot be attributed to differences in amount or composition of the fat. The contribution of the milk bile salt-dependent lipase(8, 20–22) to gastric lipolysis is probably minimal or nil, because the enzyme requires high bile salt concentrations (≥10 mM) even in the presence of products of lipolysis(51–52) as well as a much higher pH (8.0-9.0) than that of the stomach, for its activity. Furthermore, similar rates of gastric lipolysis were reported in one infant fed pasteurized milk, a process that would have inactivated the milk lipase(53).

The more efficient gastric digestion of the fat in human milk might be related to the structure and size of the fat particles in milk compared with formula. Milk fat globules have a diameter of 4.0 μm(54, 55), and their structure is characterized by a triglyceride core and a globule membrane composed of phospholipid, cholesterol, and protein(55), whereas formula fat particles have a smaller diameter (0.6 μm for SMA SP), and the triglyceride core is surrounded by phospholipids. It is possible that gastric lipase is able to access the core triglyceride more easily in milk fat globules and that excess phospholipid is an obstacle to the access of the enzyme to formula triglyceride. Phospholipids are a major barrier to triglyceride hydrolysis by milk bile salt dependent lipase(51, 52), and a mixture of proteins and phospholipids prevent triglyceride hydrolysis by pancreatic colipase dependent lipase(7). Indeed, the hydrolysis of milk fat globule triglyceride by either of the above enzymes depends upon the initial predigestion by gastric lipase(8–10).

Electron microscopy of the fat particles at the end of 50 min of gastric digestion shows that the particles maintain their initial shape and that the products of lipolysis are contained within the particles (Fig. 6)(56). Similar milk fat globule-contained lipolysis products were previously reported during in vitro incubation of milk fat globules with lingual lipase (an enzyme identical to gastric lipase) and visualization by phase contrast or freeze etching techniques(56).

In line with the significantly greater gastric lipolysis of human milk fat than of formula fat, fat absorption was about 12% higher in milk-fed than in formula-fed infants. In addition, this study confirms earlier reports of similar rates of fat absorption in infants fed LCT- or MCT-containing formulas(14, 26) and shows that the process of fat digestion and absorption is well developed in premature infants.

Based on in vitro studies it has been suggested that the main function of gastric lipase is to initiate fat digestion and thereby facilitate the subsequent action of milk bile salt dependent lipase and pancreatic colipase dependent lipase(8). The data presented in this study show that gastric lipolysis is much more extensive in vivo than in vitro and that this is probably the reason for the excellent fat absorption in formula fed infants who do not benefit from the action of milk bile salt dependent lipase. In conclusion, the present study demonstrates that differences in type of feeding: different fatty acid profiles (LCT or MCT), different emulsions (natural or artificial) and different fat particle sizes do not affect the level of gastric enzymes either in the fasting state or postprandially. This study also shows that while gastric lipase output is similar in premature infants to that of adults on a high fat diet, postprandial pepsin output is markedly lower in preterm infants than in adults. This study suggests that the contribution of gastric lipase to overall fat digestion might be greater during physiologic pancreatic insufficiency (in the newborn) or pathologic pancreatic insufficiency [cystic fibrosis(57, 58) and chronic alcoholism(59)] than in healthy adults(60, 61).