Between 16–40% of the infants with constipation experience symptoms before the age of six months 123. In approximately 90% of infants no specific organic cause can be found 4. It is well established that the bowel pattern in infants is influenced by the type of feeding in the first months after birth. Constipation is more commonly found in formula-fed infants, who have a greater tendency to produce hard stools compared to breast-fed infants 5. Differences in the composition between breast- and formula feeding may explain this finding.

The structure of lipids differs between human milk and infant formulas. In both human milk and infant formulas palmitic acid is the predominant saturated fatty acid. In human milk 70–85% of palmitic acid is positioned at the sn-2 position of the triacylglycerol molecule, whereas in regular infant formulas 88–94% of palmitic acid is found at the sn-1 and sn-3 position 678910. Lipolysis of triacylglycerol by pancreatic lipase occurs predominantly at the sn-1 and sn-3 positions, yielding free fatty acids and a 2-monoacylglycerol 1112. Subsequently, free palmitic acid may form insoluble calcium fatty acid soaps which are excreted via the feces, resulting in firmer stools. Stool hardness has been positively associated with the presence of calcium fatty acid soaps in the stools 5. In human milk however, palmitic acid esterified at the sn-2 position of the triacylglycerol molecule is well absorbed as 2-monopalmitin, since it readily forms mixed micelles with bile acids 11131415.

Human milk is further known to be a rich source of oligosaccharides 16. These oligosaccharides resist digestion in the small intestine and thus reach the colon unaltered, where they serve as prebiotics 17. They act as growth substrate for bifidobacteria, which are thought to have beneficial effects on the host's health by supporting the gut barrier, stimulating normal intestinal function, and strengthening the immune system 181920. In addition, due to their non-digestibility, they may be considered to be a form of soluble fibres and contribute to the softer stools produced by breast-fed infants 1721.

Based on these findings, the concept of adding modified triacylglycerol and prebiotic oligosaccharides to infant formulas has arisen. A new infant formula (NF; Nutrilon Omneo, Nutricia Nederland BV, Zoetermeer, the Netherlands) was developed which contains modified vegetable oil with a high proportion (41%) of palmitic acid at the sn-2 position, a mixture of prebiotic oligosaccharides, partially hydrolyzed whey protein and a reduced lactose content. The oligosaccharides mixture consists of 90% short-chain galacto-oligosaccharides (GOS) and 10% long-chain fructo-oligosaccharides (lcFOS), 0.8 g/100 ml, and resembles human milk oligosaccharides with respect to its molecular weight distribution and high galactose content 22. The effect of NF on stool frequency and consistency has been assessed in one study in healthy term infants 23. Infants receiving NF were found to produce softer stools than those fed a regular infant formula. We hypothesized that this NF will also have a positive effect stool characteristics in constipated infants.

To our knowledge this is the first double-blind cross-over study evaluating the effect of Nutrilon Omneo (NF) on stool characteristics in infants with constipation. Our data show that constipated infants with hard stools fed NF, containing a high proportion of sn-2 palmitic acid and a mixture of prebiotic oligosaccharides, improved more often to softer stools compared to whey-based SF-fed infants. Defecation frequency increased significantly compared to baseline in both groups, but was not different between the NF-fed and SF-fed infants. The formula was well tolerated and growth rates were similar on both formulas.

Previous studies in healthy infants revealed a wide variability in defecation frequency depending on age and the type of feeding 212425. Fontana et al. showed a decline in defecation frequency from an average of 3 stools per day in the first month of life to 1.4 per day at 3 years of age 24. Furthermore, breast-fed infants have a defecation frequency twice as high as formula-fed infants in the first 12 weeks of life 21. In this study in infants with constipation, the mean defecation frequency at enrollment was 3.5 times per week. A total of 45% of all infants presented with a defecation frequency of < 3 times/week. Two recent follow-up studies in constipated infants found different defecation frequencies at enrollment of 6.5 times per week and 2 times per week, respectively 2627. This disparity is most likely explained by the difference in patient populations. The study by Loening-Baucke et al. and our study were conducted in infants referred to general pediatric out-patient clinics. In the study by Van den Berg et al. only infants with severe functional constipation that required referral to a specialized clinic to rule out Hirschsprung's disease were included. The difference in age distribution between the studies may also have contributed to the difference in outcome, since we included younger infants up to 5 months of age, but the other two studies included children up to a maximum of 2 years of age 2627.

In comparison to these recent follow-up studies in constipated infants, we found a higher percentage of infants with painful defecation (82%). Hard stool consistency in more than half of the infants just partially explains this finding, since the presence of painful defecation did not improve in accordance with a softer stool consistency. Loening-Baucke et al. reported hard stool consistency in 93% of all constipated children and painful defecation in only 41%. Van de Berg et al. did not report on stool consistency, but painful defecation was present in 49% of the infants. Differences may be explained by the fact that painful defecation at this young age is a very subjective measurement and difficult to objectify by a parent on a daily basis. The fact that this study was a multi-centre trial may also have lead to an inter-observer difference in the registration of changes in painful defecation.

In our analysis infants receiving NF showed more improvement of hard stools to soft stools than those fed SF, although significance was only reached in infants completing the cross-over study. This result is in line with the findings of two previous studies demonstrating softer stools in healthy term infants fed high sn-2 palmitate formula 915. This effect is attributed to a reduced fecal excretion of calcium-fatty acid soaps 528. Another explanation for the difference in stool consistency is most likely the addition of a prebiotic mixture of GOS/lcFOS in the Nutrilon Omneo formula. Earlier studies in healthy infants have shown that supplementation of an infant formula with this mixture resulted in an increased number of fecal Bifidobacteria and softer stool consistency 232930. Moro et al. found that this effect was dose-dependent and softer stools were comparable to stools of breast-fed infants at a level of 0.8 g/100 ml of GOS/FOS mixture 30. Similar to our results, Schmelzle et al. showed softer stools, but no statistical difference in defecation frequency in healthy term infants fed NF 23. This positive effect on stool consistency is of clinical importance in our patient population, as a majority of infants (61%) presented with hard stool consistency as main symptom of their constipation.

Additionally, previous studies have shown that formulas containing hydrolyzed protein can produce softer stools 31. Therefore, the presence of hydrolyzed whey protein may also have contributed to the stool softening effect of NF. In this respect, the addition of some hydrolyzed protein (about 25% of total protein) to SF may explain the observed increase of defecation frequency on both formulas. However, the observed increase in defecation frequency was stronger in infants fed NF than those fed SF, but no significant difference was found.

A limitation of this study is that more than one third of the constipated infants enrolled did not complete the study protocol. Due to this high drop-out rate, no cross-over data was available from these children resulting in loss of power in our analysis. Therefore our findings on defecation frequency and stool consistency need to be confirmed in a larger clinical trial in order to gain more insight into the effects of the composition of this NF.

This drop-out rate is of further concern because it may potentially introduce a bias. However, besides a significantly lower age at enrollment in the drop-out group, baseline characteristics showed no difference between the infants who dropped out and the remaining infants. There was a relationship between the drop-out rate and the feeding type, since the most important reasons for drop-out were: 1) a significant improvement after the first treatment period on NF, or 2) the recurrence of symptoms of constipation after the switch to SF in the second period. Understandably, parents refused to continue the study when their infant had improved or when symptoms of constipation recurred. In general, parents of sick children have emotional and ethical problems to accept the risk of recurrence of the initial symptoms. The latter is probably the reason for the lack of scientific data evaluating the effect of infant formulas or oral laxatives in infants with constipation.

In conclusion, this study demonstrates that the use of an infant formula with a high proportion of sn-2 palmitate, a mixture of prebiotic galacto-oligosaccharides and fructo-oligosaccharides and partially hydrolyzed whey protein may lead to softer stools in constipated infants. Thus, constipated infants who present with hard stools may benefit from a change form SF to this NF as a first treatment step, but larger randomized clinical trials on the efficacy of this new formula are needed.

This study was supported by a grant of Nutricia Nederland BV, Zoetermeer, The Netherlands.

FdL contributed substantial to conception and design of the study and collection of the data. MIG contributed to the acquisition of data and has been involved in revising the manuscript. JBR contributed significantly to the statistical analysis. MEB carried out the statistical analysis and wrote the manuscript. JAT helped to critically revise the manuscript. MAB supervised the design and coordination of the study and helped to draft and revise the manuscript. All authors read and approved the final manuscript.