Wednesday, June 11, 2014

Might the gut explain colic?



Infant colic is a common problem among infants, with between 10-30% of US infants identified as colicky infants. Despite the common experience that is colic, colic is in fact poorly understood. Medically, colic is defined using Wessel’s criteria, perhaps better known as “The Rule of 3s”: crying that last more than 3 hours a day, for more than 3 days a week, for over 3 weeks, although most parents do not seek medical advice for colic.  There are also stereotypical behaviors associated with a bout of colic – the infant will pull their legs up as if in pain, there is increased abdominal bloating, passing of gas, face flushing, and a specific, high pitched cry.  Colic usually resolves by 4 months, making it difficult to determine if treatments worked, or the colic naturally resolved on its own. 

Historically, any number of factors have thought to be play a role in the development of colic ranging from the tired old trope of the refrigerator mother (cold, uncaring mother), to reflux, protein sensitivity, or issues with lactose digestion. 

Emerging evidence does suggest that some of the cases of colic may be caused by infant reflux, GERD, or protein sensitivity. However, in a large number of infants, these causative factors can be ruled out. New evidence however, suggests that the factors underlying colic may be quite complex – and definitely non-human. Colic, it seems, may be a behavioral response by infants to differences in the microorganisms living in their GI tracts.

Three studies have investigated the association between infant gastrointestinal microbiome and infant colic, with some very interesting results.
For the first study, Savino et al., (2004) collected fecal samples from 71 infants aged 15-60 days with no prior use of antibiotics or probiotics. There were no differences in the amount of several common aerobic GI bacteria. However, infants with colic were much less likely to have bacteria from the genus Lactobacillus. Those colicky infants that did have Lactobacillus had much smaller colony forming units (a way of quantifying the amount of bacteria in the infant’s GI tract) 1.26 cfu per gram, compared to 2.89 cfu/gram.

Figure 1: Lactobacillus acidophilus at high resolution.  This is one of the more common forms of Lactobacillus, and is frequently used in the production of yogurt. Image: http://microbiologyfall2010.wikispaces.com/Probiotics+-+Do+you+want+some+bacteria+with+that%3F
In the follow-up study, Savino et al., (2005) investigated these microflora differences in 30 colicky and 26 unaffected controls; all exclusively breastfed. Colic was defined by physician assessment, and fecal samples were collected from each infant.  Fecal samples were analyzed for bacterial type – down to the species level.  Unlike the prior study, Savino et al., reported no differences in total Lactobacillus colony forming units between the colicky and non-colicky infants. However, at the species level, there were some striking differences.

None of the colicky infants had Lactobacillus acidophilus. No non-colicky (control) infants had Lactobacillus brevis or Lactobacillus lactis lactis. These differences may be tremendously important in influencing infant health and GI function. First, while all Lactobacillus are anaerobic bacteria (they do not require oxygen), a few species are capable of glucose fermentation, producing as byproducts carbon dioxide and ethyl alcohol. The species that produce carbon dioxide and ethyl alcohol? You guessed it: Lactobacillus brevis and Lactobacillus lactis lactis – the very bacteria ONLY present in colicky infants.

Lactobacillus acidophilus (Figure 1), the bacteria not found in colicky infants, is an important contributor to immune function in the GI system, and likely promotes immune activity and the development of oral tolerance to food antigens (reduces risk of reactions to foods). Presence and absence here becomes a perfect storm: the increase in gas and ethyl alcohol producing species and the loss of protective species may increase the risk of GI infection in colicky infants, and may contribute to the gas and distress commonly associated with colic. This may also explain why fecal calprotectin, a hormonal marker of inflammation commonly used as a measure of GI damage, is increased in the stools of infants with colic (Rhoads et al., 2009).

However, these three studies are somewhat limited in their study design – primarily by the use of a single sample per infant. Infant microflora may differ between colicky and non-colicky infants – but how does this process occur?

deWeerth et al., (2013) have some answers. Piggy-backing on an existing study, they identified 12 infants with colic and 12 infant without colic from a larger sample of infants. The infants had similar ages, birth weights, and current weights, but differed in the amount of crying reported by the parents. They then analyzed nine fecal samples collected from day 2 to 4 months postpartum, using DNA measurements to determine the types of bacteria in gut microflora. As found in the earlier studies, there were significant differences in the types of bacteria, especially Lactobacillus. These differences were present in the samples collected at 2 weeks postpartum - colicky babies already had less Bacteroidetes, and more E. coli and Enterobacteria, while non-colicky babies had more Bifidobacteria and Lactobacillus gasseri. Most striking however, was the decreased diversity in bacterial species found on days 14 and 28 in the feces of the future colicky infants - and remember, these samples were collected before the colic emerged. 


Figure 2: Image from deWeerth et al., 2013. The distribution of bacterial groupings in the fecal samples collected from infants at day 14 (before the onset of colic) classified by whether or not the infant developed colic. Each Circle with a letter is a non-colicky infant, each red square with a letter is a colicky infants. There is little overlap in the bacterial groupings of the infants. The paper is open access and you can read it here
By 4 months postpartum, there were no differences in the microflora between colicky and non-colicky infants. This is about the time that colic usually resolves, and deWeerth et al., speculate that this shift in the microbiome may be one of the potential mechanisms.

Here is what I want to know : if there are known differences in the GI microflora between colicky and non-colicky infants, what factors contribute to these differences? deWeerth speculates genetics or chance encounters may contribute to these differences. But what is if is something else? What is the differences start with milk? Is it possible that differences in the oligosaccharides in milk may promote the growth of different types of bacteria? Alternatively, may the cfu units in milk differ between women? If the later, could maternal probiotics be a treatment for colic? No one really knows – as far as I can tell, no one has investigated milk composition and microflora differences in the milk or guts (and maybe vaginas?) of mothers who have infants with or without colic. Or, perhaps following the elegant study design of deWeerth, a longitudinal study utilizing maternal and infant microflora measurements of control and colicky infants recruited into the study at birth and followed over the first four months of life. In any event, there are a lot of questions and missing pieces remaining – and milk may be an important one!

Next month: the milk microbiome: or there are bacteria in human milk and that is a good thing.

References

Rhoads JM1, Fatheree NY, Norori J, Liu Y, Lucke JF, Tyson JE, Ferris MJ. Altered fecal microflora and increased fecal calprotectin in infants with colic. J Pediatr. 2009 Dec;155(6):823-828.e1. doi: 10.1016/j.jpeds.2009.05.012.


Savino F, Bailo E, Oggero R, Tullio V, Roana J, Carlone N, Cuffini AM, Silvestro L. Bacterial counts of intestinal Lactobacillus species in infants with colic. Pediatr Allergy 
Immunol. 2005 Feb;16(1):72-5.


Savino F, Cresi F, Pautasso S, Palumeri E, Tullio V, Roana J, Silvestro L, Oggero R. Intestinal microflora in breastfed colicky and non-colicky infants. Acta Paediatr. 2004 Jun;93(6):825-9.


de Weerth C, Fuentes S, Puylaert P, de Vos WM. Intestinal microbiota of infants with colic: development and specific signatures. Pediatrics. 2013 Feb;131(2):e550-8. doi: 10.1542/peds.2012-1449.


Monday, March 17, 2014

Mother’s milk/Baby’s Water: Selective pressures of infant hydration on milk composition and infant feeding structure in human populations



Human milk is, on average, approximately 85-90% water (Hinde and Milligan, 2011), reflecting our long evolutionary history as primates with dilute, high sugar milks. The reference composition data for milk describes milk as approximately 4.0% fat, 1.2% protein, and 7.2% sugar, although both individuals and populations will vary in the distribution of these macronutrients.

Human milk is considered dilute when compared to that of other mammals. The best interpretation of this comes from the work of Ben Shaul (1962) who described humans as a high contact, high frequency nursing species. Other primates, with a few exceptions, also have dilute milks (Hinde and Milligan, 2011).  As with humans, the dilute composition of primate milk is thought to be driven by distinct patterns of infant care – high levels of contact, high nursing frequency, and low volume transfer per bout. Environmental factors, such as dry and arid environments and elevated rates of whole body water turnover, also predict dilute milks (an example would be camels).

However, within human populations, much less is known about the ways in which the environment may influence the composition of milk, particularly the amount of water in the milk. For many species, geographical ranges are quite limited, and there is little variation in ecological stressors such as temperature or aridity.  Humans are among a small number of primates with a wide geographical distribution, and occupy a diversity of environments. These environments present any number of ecological stressors (temperature, aridity, altitude, humidity, pathogens) that may influence breastfeeding behaviors or milk composition.

Human infants are at increased risk of dehydration compared to older children and adults. Infants have higher whole body water turnover rates and immature kidney function, limiting their capacity to reserve water should intake decrease or water loss (often associated with diarrhea) increase.  However, the majority of discussions on milk composition and breastfeeding behaviors have largely ignored maintaining infant hydration as a possible selective pressure (Bentley 1998). Is it possible that hydration has been an important factor in shaping breastfeeding behaviors, and maybe even some aspects of milk variation, in humans?

“We may conjecture that at different climatic extremes different nutritional priorities are placed upon breastfeeding. So in tropical or arid conditions, one might presume that high evaporative water loss would place the emphasis on maximizing water throughput to prevent dehydration.” – Michael Woodridge (1995).

Looking at population diversity in nursing frequency among traditional foraging societies, Woolridge (1995) reports increased nursing frequency among mothers living in arid or semi-arid environments. One of the best examples of this are the !Kung, a population of foragers living in the Kalahari desert (Figure 1). The !Kung have very high nursing frequencies – on the order of 4 times an hour! Other foraging populations living in arid or semi-arid conditions also show increased nursing frequency, although there appears to be variation within these climate extremes.

Infants who feed with a high frequency but short duration may be biologically altering the composition of the milk. Daly et al., (1993) and more recently Kent et al., (2006) have shown that the amount of fat in milk (they did not measure milk lactose) changes based on the frequency of feeding. More frequent feeds predict lower overall change from the end to the beginning of the next feed, and closely spaced feeds may have an overall effect of maximizing water transfer and reducing the amount of fat in the milk. The infant must nurse more frequently to meet energy and fat needs and may, over the course of the day, take a large quantity of milk. 

Biologically, this might be incredibly important in thinking about the ways in which differences in milk composition are often interpreted as differences in milk quality, especially in older published studies. Discussions of human milk quality are often synonymous with energy and fat, completely ignoring the fact that in certain environments where dehydration is a risk and total body water turnover rates high, low fat milk with a high water content might be adaptive. This milk will keep the infant drinking regularly, and may be important for maintaining physiological well-being and preventing dehydration. As discussed above, total daily fat intake by the infant should be equivalent to that reported in other populations because of the overall increase in milk volume consumed by the infant. However, if we were to simply look at the milk composition without the added context of nursing frequency or total volume of milk consumed over the course of the day (and these are separate issues), it might appear that the infant was not getting enough to eat. 

Figure 1: A Bedouin mother struggles with a tent and a baby. Image:AFP, by way of http://womennewsnetwork.net/2012/07/03/rising-number-bedouin-women-enter-work-force/.


One other interesting piece of support for this dehydration hypothesis comes from studies looking at milk composition across seasons, comparing summer and winter, or rainy and dry seasons for example. Few studies are available, and observations are confounded by the fact that in many tropical and sub-tropical populations, seasonal workloads and pathogen risk will vary greatly. Yagil (1986), in a sample of Bedouin mothers, reported a modest increase (+4%) in the percentage of water in milk during the summer compared to the winter (and a decrease in milk fat), although the collection methodology was somewhat problematic. 

While the case is far from solved – and really, the population level data on aridity and breastfeeding frequency may not exist – it remains an interesting hypothesis for some of the possible variation in milk composition and breastfeeding behaviors. Moreover, it may be important to think about changes to milk composition across seasons – is milk more dilute during hot or arid months, promoting increased nursing to maintain hydration? Is some of the regional variation in human milk composition and nursing frequency driven not by hunger but by thirst?

References
Bentley, G.R. (1998), Hydration as a limiting factor in lactation. Am. J. Hum. Biol., 10: 151–161. doi: 10.1002/(SICI)1520-6300(1998)10:2<151::AID-AJHB2>3.0.CO;2-O

Daly SE, Di Rosso A, Owens RA, Hartmann PE. (1993) Degree of breast emptying explains changes in the fat content, but not fatty acid composition, of human milk. Exp Physiol. 78(6):741-55.

Hinde, K. and Milligan, L. A. (2011), Primate milk: Proximate mechanisms and ultimate perspectives. Evol. Anthropol., 20: 9–23. doi: 10.1002/evan.20289

Kent JC. (2007) How breastfeeding works.J Midwifery Womens Health. 52(6):564-70.

WoolridgeM(1995) Baby-controlled breastfeeding: Biocultural implications. In P Stuart-Macadam and KA Dettwyler (eds.): Breastfeeding: Biocultural Perspectives. New York: de Gruyter, pp. 217–242.

Yagil R, Amir H, Abu-Rabiya Y, Etzion Z (1986) Dilution of milk: A physiological adaptation of 
mammals to water stress. J. Arid Environ. 11:243–247.