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Human milk fortifiers (HMF) are commonly added to human milk to increase caloric density and nutrient delivery to preterm infants. TGFβ2 is present in human milk, has immunoregulatory properties, and is involved in growth, differentiation, and repair of neonatal gut epithelia. The effect of HMF on TGFβ2 concentration is unknown. We sought to measure the concentration of TGFβ2 in whole milk following the addition of a commercially available powdered HMF. Human milk samples (N=23) were collected and stored at -20°C. For analysis, milk samples were thawed and divided into two aliquots. HMF was added at a concentration of 0.0384 g/mL (or one 0.96 g packet per 25 mL) to the 2nd aliquot of each pair. Controls included:(1) HMF at a concentration of 0.0384 g/mL in calibrator diluent (CD) and (2) HMF 0.0384 g/mL in CD with recombinant TGFβ2 at 2000 and 500 pg/mL. The concentration of the 25 kD acid-activated TGFβ2 isoform was measured using an ELISA (R&D Systems). The mean±SD concentration of TGFβ2 in unfortified milk was 5554±5345 (range 143-17640) pg/mL human milk. The mean±SD concentration of TGFβ2 in fortified milk was 7135±4774 (range 1641-17390) pg/mL. The mean relative increase in milk TGFβ2 concentration after the addition of HMF was 3.4 times that of unfortified milk. The mean TGFβ2 concentration in HMF control was 2518 pg/mL. HMF doubled the concentration of TGFTGFβ2 standards. In summary, with the addition of HMF to whole milk samples, TGFβ2 concentration decreased 2-to-10% in 4 samples but increased 1-to- 3174% in 19 samples. It appears that HMF is an additional source of TGFβ2 when added to human milk. What effect this alteration in human milk TGFβ2 concentration has on neonatal immune and gut function remains unknown.

The Effect of Antenatal Steroids on Transforming Growth Factor β-2(TGFβ 2 ) Concentrations in Human Milk ♦ 588

Human milk contains transforming growth factor-beta isoforms(TGFβ1 and β2). TGFβ affects growth and differentiation of neonatal intestinal epithelia, and modulates restitution of the gut epithilium following injury. A differential distribution of some bioactive factors in milk fat and aqueous fractions has been shown, which could affect bioavailability. It is unknown if TGF-β2 shares similar distribution properties. We sought to determine if there is a differential concentration of TGFβ2 in human milk aqueous and fat compartments. Eighteen milk samples were collected from 15 lactating mothers on postpartum day 2 to 27; gestational age of their infants at delivery ranged from 24 to 40 weeks. Whole milk was centrifuged at 2500g × 20 min at 4°C to achieve separation and isolation of its fat, aqueous and cellular components. Sodium taurcholate(STC) was added to the fat for emulsion. The concentration of acid-activated TGFβ2 in the aqueous and fat components of human milk was measured by ELISA (RD sensitivity 55 pg/ml sample; cross-reactivity with other TGFβ species/receptors is not significant; standard curve unaffected by STC). TGFβ2 was detected in all aqueous samples (6214 ± 5083 pg/mL; range 1619 to 20686); and all fat samples (6987 ± 5344 pg/mL; range 1736 to 20576). Similac Special Care formula and Enfamil Premature formula, also tested, did not have detectable levels of TGFβ2. In conclusion TGFβ2 is found in high concentrations in both the aqueous and fat compartments of human milk. These findings may be of significance for differential distribution and bioavailability of TGFβ2 in the newborn gut. Results of this study are important to future research, as fat is often discarded during human milk investigations.

Human milk contains many cytokines, including transforming growth factor-beta isoforms (TGFβ1 and β2). TGFβ appears to control growth and differentiation of neonatal intestinal epithelia, and to act in the repair of the gut following injury. It is thought that the bioavailability of these factors can be affected by their compartmentalization in milk. While an increase of certain bioactive factors within the fat compartment of milk has been shown, it is unknown if TGFβ1 shares similar distribution properties. We sought to determine if there is a differential concentration of TGFβ1 in the human milk fat layer compared with whole milk and its aqueous fraction. Thirty-six whole milk samples and an additional 13 milk fat samples were collected from 35 lactating mothers on postpartum day 3 to 6 mo.; gestational age of their infants at delivery ranged from 32 to 42 weeks. Whole milk was centrifuged at 1000g× 20 min at 4°C to achieve separation and isolation of its fat, aqueous and cellular components. Tris buffered saline (TBS; 200 μL) with 10μL Tween-20 was added to the fat for emulsion. The concentration of acid-activated TGFβ1 in whole milk, and its aqueous and fat components was measured by ELISA (Promega; sensitivity 25 pg/ml sample; cross-reactivity with other TGFβ species of