Earlier studies showed that the milk composition of term mothers changes significantly during introduction of complementary feeding [5-8]. The objective of this pilot study was to describe the change in concentration of protein, triglycerides, lactose, sodium, potassium, calcium and phosphorus in preterm milk until introduction of other feeds at 6 months.

This longitudinal study was conducted in a neonatal intensive care unit in Northern India after obtaining approval from Institutional ethics committee.

Mothers of inborn singleton preterm neonates (gestation <34 weeks) were enrolled – after obtaining informed consent – if they were feeding breast milk (directly or expressed). Mothers who started other milk or feeds before 4 months of postnatal age were excluded. Mothers were counseled by a nurse certified in lactation counseling. The clinical profile of the mothers and infants was recorded.

Breast milk samples were collected at 7, 28, 90 and 180 days of life, with a margin of ± 2 days. All samples were collected between 2-4 PM; 5 mL milk was manually expressed into wide-mouthed, acid-washed, freezer-safe, plastic vials after the infant had nursed for 3 minutes, or after expressing for 3 minutes, whichever was applicable [5]. The plastic vials were stored at -20°C until analysis. Maximum duration of storage until analysis was 4 months. Proteins were analyzed by micro-Kjeldahl method, triglycerides by Roese-Gottlib method, lactose by enzymatic hydrolysis; sodium, potassium and calcium by ion selective electrodes and phosphorus by ammonium molybdate method [9-11]. Triglycerides were assayed as a surrogate for milk fat content as 98% of breast milk fat is triglycerides [12].

A sample size of convenience was enrolled over one year. Repeated measurements were analyzed by mixed linear models. Normally distributed numerical variables were compared by Students t-test and skewed variables by Mann-Whitney U test.

The lactose concentration increased with postnatal age until day 90; and then declined by day 180 (Table I ). On repeated measures analysis, the triglyceride concentration showed a significant increase with postnatal age until day 180 (P= 0.001). The protein concentration showed a statistically significant decrease with postnatal age (P<0.001). On day 180, sodium concentration was significantly higher in the milk of mothers who had introduced mixed feeding (P=0.02) (Table II).

TABLE I  Change in Composition of Preterm Human Milk over First 6 Months of Life

TABLE II	Milk Composition In Mothers Exclusively Breastfeeding and Those Giving Mixed Feeding by 6 Months Postnatal Age

We demonstrated a progressive increase in triglycerides and sodium, a progressive decline in protein, and non-significant changes in other nutrients in preterm human milk from 7 to 180 days postnatal age. The milk of preterm mothers, who had introduced mixed feeding after 4 months, had a higher content of triglycerides and sodium.

Overall, these changes are similar to those reported for term mothers who had introduced complementary feeding. Neville, et al. [8] reported a significant increase in sodium and protein, and decrease in lactose concentration when the milk output of term mothers fell below 400 mL/day. Garza, et al. [7] reported higher protein and sodium levels after introduction of complementary feeding in term-born infants.

The change in composition of preterm human milk during introduction of mixed or complementary feeding may be a result of breast involution and increased inter-cellular permeability [5,8,13,14]. Lack of exclusive breast milk feeding causes milk stasis – an important stimulus for opening of tight junctions and increasing permeability. The change in composition may provide a survival advantage to the infant.

The limitations of the study included a small sample size, loss of patients during follow-up, inability to draw a milk sample representative of the entire breast-feed, and lack of anthropometric data on the infants at 6 months and its correlation with breast milk composition. Storage of milk in the freezer compartment up to a maximum of 4 months may have altered the composition of the milk samples to variable extents.

We conclude that the composition of preterm human milk changes around the period of introduction of mixed feeding at about 6 months. If these preliminary findings are confirmed in larger studies it would imply that akin to colostrum – breast milk composition changes according to the needs of an infant who is started on mixed feeding.

Acknowledgements: Lactation consultants Mrs Rama Mahajan and Mrs Uma Arora.

Contributors: SD: planned the study, collected the samples, analyzed the data and finalised the manuscript; SS: analyzed the data and drafted of the manuscript; RP: analyzed milk samples.

Funding: PGI intramural Research Scheme;.

Competing interests: None stated.