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Motor activity is responsible for the aboral movement of nutrients and, as such, regulates gastric emptying and intestinal transit in adults. During fasting the adult intestine exhibits the interdigestive cycle, as motor quiescence is replaced by irregular activity that increases in intensity and occurrence until it is replaced, in turn, by intense phasic contractions migrating distally through the intestine. This latter event, called the MMC, is responsible for the forward propagation of nutrients and digestive juices through the gut(1). The absence of MMCs is associated with slow intestinal transit and feeding intolerance in adults who have diabetic gastroparesis as well as other neural and myogenic abnormalities(2). We have recently characterized motor patterns in preterm and term infants(3). Compared with adults, preterm infants display an absence of MMCs, prominent nonmigrating clustered phasic activity, and a paucity of motor quiescence(4). This “immaturity” of motor activity is reflected by delayed gastroanal transit of 40-75 h(5). As in adults, feeding problems in neonates are also associated with the presence of specific age-related abnormalities of motor activity(6).

Two hormones appear to modulate the initiation and/or the distal propagation of the MMC. Motilin, a 22-amino acid peptide, is produced by the enterochromaffin cells of the duodenal and jejunal mucosa and is released in cyclical manner concurrently with the occurrence of MMCs; that is, plasma concentrations increase acutely and transiently by 2-4-fold concurrently with the onset of the MMC(7, 8). Infusion of exogenous motilin induces the premature appearance of the MMC in dogs as well as humans(9–11), and infusion of a motilin antagonist suppresses the occurrence of MMCs(12). In a manner similar to motilin, plasma concentrations of pancreatic polypeptide also cycle in concert with the appearance of the MMC in the dog, pig, and man(13, 14). However, the role of pancreatic polypeptide in regulating the initiation or the propagation of the MMC in the human has not yet been confirmed(11). The antibiotic erythromycin competitively binds to the motilin receptor(15) and induces the appearance of migrating activity in dogs and humans(11, 16). Because of this, it is a potent motilin agonist commonly used as a prokinetic agent and to assess indirectly and pharmacologically the functional presence of the motilin receptor.

In the human neonate, the MMC is rarely present before 32 wk of gestational age, and it is only occasionally present in term infants(3, 4, 17, 18). We and others have shown that plasma concentrations of pancreatic polypeptide are lower in neonates than adults, but that plasma concentrations of motilin are similar in neonates and adults(19–21). In adults plasma concentrations of both of these peptides increase 2-4-fold in a cyclical fashion with the onset of migrating activity(7, 8, 14). We postulated that the paucity of MMCs in the preterm infant may be due to the absence of hormonal cycling of motilin and/or pancreatic polypeptide, or alternatively, due to the absence of the motilin receptor.

Therefore, the specific aims of these two studies were to determine whether plasma concentrations of motilin and pancreatic polypeptide surge with the occurrence of the MMC in term neonates and to use erythromycin to indirectly assess whether the motilin receptor is present in preterm infants before and after 32 wk of gestation.

METHODS

Population and study design. Study A. In study A, subjects were nine term infants born at 37-42 wk of gestational age who were admitted to the Neonatal Intensive Care Unit at Texas Children's Hospital or Ben Taub General Hospital for ventilator support for hyaline membrane disease or pneumonia (Table 1). Each infant had an indwelling vascular catheter and an orogastric tube present for routine care in the Neonatal Intensive Care Unit. Infants who had congenital anomalies or who were receiving opiates were excluded from the study. A single study was performed when the infant had recovered from its need for ventilator support and the attending neonatologist had ordered that feedings be initiated. Most infants achieved these goals by postnatal d 7. This study was approved by the Institutional Review Boards of the two participating hospitals, and each infant's parent(s) provided consent for the infant's participation in this study.

Table 1 Patient characteristics
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The association of plasma motilin and pancreatic polypeptide with nonmigrating and migrating motor activity was studied in these infants. None of these infants had been enterally fed at study entry; thus all were fasting. Motor activity was recorded for 4-6 h, and the recording was visually monitored for the presence or absence of MMCs. An MMC was defined to be present if phasic motor activity sustained for more than 2 min migrated across three or more leads, as previously defined for infants(3, 4). Only episodes that were initiated in the antrum and migrated to the duodenum were defined to be MMCs. Two 1.0-mL blood samples were drawn when migrating activity was absent, and two more were withdrawn within 1 min of the visual recognition of an MMC(Fig. 1, A and B, respectively). In an attempt to limit the volume of blood required for sampling and to decrease the lag time of blood sampling with respect to the initiation of the MMC, the manometric recording was constantly monitored at the bedside. If a series of phasic contractions occurred in the antrum, a blood sample was withdrawn from the catheter. If the subsequent recording displayed that the phasic activity then migrated distally to the duodental ports, the sample was placed on ice. If there was no subsequent migrating activity noted, the blood was reinfused so that blood sampling could be attempted again later in the recording.

Figure 1
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(A) A representative manometric recording depicting nonmigrating activity in a term infant. Motor activity recorded in the antrum is shown in the top line, activity in the antropyloric junction in the second, and duodenum in the third and fourth. The arrow indicates the time when one of two blood samples was drawn for the determination of plasma motilin and pancreatic polypeptide concentrations as described in the text. (B) A representative manometric recording in the same infant shown in A, depicting motor activity that migrates distally across one antral and three duodenal leads. (The second lead has migrated from the antropyloric junction to the proximal duodenum.) The arrow indicates the point at which one of two blood samples was drawn for the determination of plasma motilin and pancreatic polypeptide concentration during the presence of MMC, as described in the text.

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Study B. Subjects who participated in this second study were 21 preterm and term infants, ranging in age from 25 to 42 wk of gestation, who were admitted to the Neonatal Intensive Care Unit at Texas Children's Hospital or Ben Taub General Hospital for prematurity, respiratory distress syndrome, congenital pneumonia, or meconium aspiration (Table 1). Entry and exclusion criteria were the same as for the first study. Of these 21 infants 14 were born with gestational ages of 32 wk or less and 7 with gestational ages greater than 32 wk. As in the first study, manometric evaluation in this second study was also performed when the baby no longer required support and the attending neonatologist had ordered that enteral feedings be initiated. As for term infants, preterm infants met these criteria by postnatal d 7.

In this study, the administration of intragastric erythromycin was used to pharmacologically assess the functional presence of motilin receptors. On the day of the study, motor activity was recorded 3 h before and 2 h after a single intragastric dose of erythromycin. Erythromycin suspension(erythromycin ethy1 succinate; 40 mg/mL; Abbott Laboratories, Abbott Park, IL) was diluted 20-fold in sterile water. Preliminary studies were performed to identify the dose needed to induce MMCs in term infants. Based on these studies, a dose of 1.5 mg/kg was administered intragastrically followed by a flush of 2 mL of sterile water to ensure complete delivery of the dose. Infants were then closely monitored for the occurrence of emesis as well as the presence of abnormalities in heart rate, heart rhythm, blood pressure, and oxygen saturation.

RIA. Blood samples were placed in cold heparinized tubes containing EDTA and transported on ice to the laboratory. Samples were then centrifuged at 1000 rpm for 10 min at 4 °C, and the resulting plasma was stored at -70 °C for later determination of motilin and pancreatic polypeptide concentration by RIA techniques previously published(22, 23). All samples were processed in duplicate as a complete set in a single assay to reduce intraassay variation.

Motilin concentrations were determined by RIA using antisera that was developed by Dr. J. C. Brown in ther guinea pig using a BSA conjugate of purified motilin isolated from porcine duodenal mucosa(22). The antisera, used at a 1:1200 dilution, has negligible cross-reactivity with gastric inhibitory polypeptide, secretin, glucagon, gastrin, cholecystokininpancreozymin, and vasoactive intestinal polypeptide at concentrations up to 10 000 pg/mL. Synthetic motilin fragments 9-22 and 13-22 do not appreciably cross-react. The 125I-motilin radiolabel was made from synthetic motilin using a chloramine-T procedure with HPLC purification. The assay standard was porcine motilin from Peninsula Laboratories (Belmont, CA, catalog no. 7171). The interassay CV was 8.0% at 99 pg/mL and 4.6% at 504 pg/mL. The intraassay CV was 11.4% at 642 pg/mL.

Pancreatic polypeptide concentrations were determined by RIA(23) using an antisera, originally developed in rabbits, at a 1:1000 dilution. The standards for human pancreatic polypeptide were obtained as a gift from Eli Lilly Company. The 125I-human pancreatic polypeptide label was made using the chloramine-T procedure with HPLC purification. The detection limit based on 2.5 SD from zero is 30 pg/mL. Cross-reactivities with peptide YY, neuropeptide Y, substance P, motilin, glucagon, gastric inhibitory peptide, and cholecystokinin 33 are all less than 0.005%. The intraassay CV was <4% at all levels, and the interassay CV varied from 8% at low levels to 12% at high concentrations.

Manometry. Motor activity was recorded using a low compliance continuous perfusion neonatal manometric system. This system, validated previously(3), provides a response rate of 57 mm Hg/s at 10 psi with an infusion rate of 0.01 mL/min/recording port. In turn, this system was connected to a 3.5-mm manometric tube. This tube was manufactured in our laboratory from polyvinyl extrusion tubing and contained four proximal manometric ports spaced 2.5 cm apart. The tube was placed in the unsedated infant who was positioned supine with the right side down. The manometric tube was perfused, and the motor activity was recorded while the tube was advanced through the stomach into the duodenum. The tube was positioned such that one manometric port was located in the antrum and three in duodenum. The final position of the tube was documented by the presence of a motility pattern characteristic of the antrum and duodenum; that is, pressure waves were occurring at 3-5 times/min and 9-11 times/min, respectively. Our previous studies have shown that, when these activities are localized at these two anatomic sites, the position of recording ports can be verified by fluoroscopy(3, 4).

Statistical and Data Analysis. In study A, the motor activity recording was visually monitored at the bedside for the appearance of MMCs. Two blood samples were drawn when no MMC was present (baseline value) and two more samples when an MMC was present. To reduce overall individual variation, the two baseline values were averaged; likewise the two MMC values were averaged. Then the averaged baseline values were compared with the averaged MMC values by paired t test.

In study B, the motor activity was reviewed qualitatively during the 3-h baseline recording and the 2-h post erythromycin recording. The presence of MMCs before and after administration of erythromycin was noted. Fisher exact test was used to compare the ability of erythromycin to induce MMCs in infants who were born at 32 wk of gestation or less to those born at 33 wk or greater.

RESULTS

Study A-Hormonal cycling. The baseline plasma concentrations for motilin ranged from 164 to 444 pg/mL. Plasma motilin values during the presence of MMCs ranged from 128 to 633 pg/mL; these plasma concentrations did not differ significantly from plasma concentrations when MMCs were not present(Table 2). Baseline plasma concentrations of pancreatic polypeptide ranged from 50 to 420 pg/mL. Plasma pancreatic polypeptide levels during the presence of MMCs ranged from 63 to 406 pg/mL; these plasma concentrations did not differ from those seen when MMCs were not present(Table 2).

Table 2 Plasma peptide concentrations in study A during the absence and presence of migrating activity (pg/ml; X ± SEM)
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Study B-Response to erythromycin. There were no MMCs noted in the baseline period among the seven infants who were at 24-32 wk of gestation. Intragastric administration of erythromycin did not induce MMCs in any of these infants (Fig. 2A and Table 3; NS). Only 3 of the 14 infants at 33-42 wk of gestation had MMCs in the baseline period, but 7 of these infants displayed MMCs within 30 min of the drug administration(Fig. 2A and Table 3; Fisher exact test p< 0.03). Five infants who had MMCs after the use of erythromycin had no MMCs during the baseline recording. One infant who had an MMC during the baseline recording failed to exhibit an MMC after erythromycin was given. Two infants had MMCs before and after the administration of intragastric erythromycin. However, the MMCs induced by the administration of erythromycin occurred prematurely in both of these infants. The periodicities of MMCs in the baseline recordings of these infants were 200 and 180 min, respectively, and the pharmacologically induced MMCs occurred 30 and 45 min after previous MMCs, respectively, in each of these infants.

Figure 2
figure 2

(A) A representative manometric recording in an infant at 26 wk of gestation 30 min after the administration of intragastric erythromycin. Although no recording was obtained from the anturm, there is no evidence of motor activity migrating distally across the duodenal leads. (B) A representative manometric recording in an infant at 33 wk of gestation 30 min after the administration of intragastric erythromycin. Phasic contractions appear in the antrum and are temporally coordinated with the occurrence of phasic activity in the three duodenal recording ports.

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Table 3 Effect of erythromycin in inducing MMCs in study B
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Characteristics of the seven pharmacologically induced MMCs were qualitatively reviewed. In all seven infants the induced MMCs occured within 30 min of the administration of erythromycin. The overall configuration of these MMCs appeared to be similar to MMCs that occurred spontaneously with respect to velocity of migration (6.7 versus 8.1 cm/min), contraction amplitude (11 versus 10 mm Hg), and duration of phase 3 in each duodenal lead (7.2 versus 7.0 min/lead).

DISCUSSION

The role of motilin in the initiation of the MMC is not clearly defined. There is an intimate temporal relationship of the hormonal cycling of motilin and/or pancreatic polypeptide and the initiation and propagation of the MMC. Thus, cyclical changes in plasma hormone concentrations may not be the sole source of regulation of the MMC; rather these cyclical changes may provide significant hormonal modulation and/or amplification of the neural regulation of the MMC(24). Despite these limitations in the current understanding of the precise mechanism of regulation of MMCs by hormonal imput, certain aspects of this modulating influence appear to be absent in the neonate. Although adult patterns of distribution of motilin throughout the intestine are established by 20 wk gestation and substantial development of the gut neuroendocrine system is completed by 25 wk of gestation, MMCs rarely occur in neonates until 32-36 wk of gestation(3, 4, 17). One explanation for the absence of MMCs in preterm infants is that hormonal cycling of motilin and/or pancreatic polypeptide is absent.

We have previously shown that fasting plasma concentrations of motilin in preterm neonates are similar to those seen in adults, whereas plasma concentrations of pancreatic polypeptide are lower(20). However, the presence of adequate “tonic” levels of motilin is insufficient to trigger migrating activity. In adult humans, the occurrence of MMCs that originate in the antrum is accompanied by the occurrence of a“surge” in plasma motilin concentrations by 2-4-fold over baseline values(7, 8). Similarly, plasma pancreatic polypeptide concentrations surge 2-4-fold with the occurrence of MMCs(14). In the current study, there was no significant change in the plasma concentrations of either of these two peptides in the neonates in concert with the occurrence of MMCs. A small population of MMCs in adults occurs independently of hormonal cycling(25, 26), and this population of MMCs appears to be present by term gestation in the human neonate, suggesting that the neural regulation of the MMC is present. We speculate, however, that the paucity of migrating contractile activity in neonates is due to the absence of the larger population of MMCs that is dependent upon surges in plasma concentrations of motilin and/or pancreatic polypeptide.

None of the infants in this study had been fed. Aynsley-Green and Shulman have shown that plasma motilin levels increase approximately 3-fold by the end of the first postnatal week in infants who have been enterally fed compared with those who have not been(27, 28). Changes in the postprandial release of motilin is not present until enteral feeds have been provided for 2-4 wk(29). We have previously shown that the administration of small enteral feedings induces the apperance of the MMC in preterm infants more prematurely than would be anticipated for their gestational age(30). Although the release of plasma motilin was not concurrently measured in these latter infants, we have recently shown that the use of small enteral feedings in hyperalimented neonatal dogs results in the premature appearance of the MMC and postnatal increases in plasma motilin concentrations(31). We speculate that the exposure of the neonatal gut to intraluminal nutrients stimulates the release of motilin and, thus, the appearance of the hormonal modulation of the MMC.

Although MMCs may occur independently of surges in motilin, the infusion of exogenous motilin can induce the premature appearance of the MMC(9, 11). For motilin to exert this biologic effect, functioning receptors must be present. Motilin receptors are present in human upper intestine(32). The administration of erythromycin, a macrolide that competitively binds to motilin receptors(15, 33), induces the premature appearance of MMCs that originate in the antrum(34). Because ethical considerations precluded the use of direct tissue binding assays to assess the presence of motilin receptors in these infants, erythromycin was used as an indirect, pharmacologic challenge to assess whether the motilin receptor was present. By using erythromycin as a pharmacologic probe, it appears from our data that the motilin receptor is functionally present in the preterm infant as early as 32 wk of gestation. This observation confirms previous reports of the identification of the MMC by manometry by 32-36 wk of gestation(3, 4, 17). In addition to its ability to induce the premature appearance of the MMC directly via motilin receptors, motilin also acts via cholinergic neural pathways, muscarinic, nicotinic, opioid, and 5HT3 receptors, as well as the release of nitric oxide(24, 35). We have previously shown that motor responses to atropine differ in preterm neonates and adults, in that gastric emptying and antral motor contractions do not decrease in response to atropine(36). Therefore, the apparent inability of erythromycin to induce MMCs in very premature infants may be due to an absence of motilin receptors. An alternative explanation is that the neural mechanisms that regulate MMCs are not yet present and therefore cannot be recruited by the motilin receptor.

The presence of motor responses to erythromycin in infants who are born with gestational age greater than 32 wk may offer the neonatologist the ability to pharmacologically enhance gastrointestinal function in these older preterm infants. Motor responses to erythromycin are dose-dependent: lower doses enhance motor activity (1-3 mg/kg), whereas higher doses cause continuous high amplitude contractions or profound motor quiescence(37). Motor responses occur approximately 20-40 min after the oral or i.v. administration of erythromycin(37). Erythromycin has been described to be an effective prokinetic agent in children(38), but randomized, prospective trials using erythromycin in preterm infants have not yet been done.