Changes in Proteases, Antiproteases, and Bioactive Proteins From Mother's Breast Milk to the Premature Infant Stomach

Abstract

Objective: Our previous studies suggested that human milk proteases begin to hydrolyze proteins in the mammary gland and continue within the term infant' stomach. No research has measured milk protease and pepsin activity in the gastric aspirates of preterm infants after human milk feeding. This study investigated how the concentrations of human milk proteases and protease inhibitors changed in the premature infant stomach.

Methods: Human milk and infant gastric samples were collected from 18 preterm-delivering mother-infant pairs (24-32 week gestational age). Paired human milk and gastric samples were collected across postnatal age (2-47 days). Protease concentrations were determined by spectrophotometric or fluorometric assays, and the concentrations of protease inhibitors and bioactive proteins were determined by enzyme-linked immunosorbent assay. Paired t tests were applied to compare enzymes, antiproteases, and bioactive proteins between human milk and gastric samples.

Results: Our study reveals that although human milk proteases, including carboxypeptidase B2, kallikrein, plasmin, cathepsin D, elastase, thrombin, and cytosol aminopeptidase, are present in the preterm infant stomach, only plasmin and cathepsin D can actively hydrolyze proteins at gastric pH. Enzyme-linked immunosorbent assay and peptidomic evidence suggest that all milk antiproteases as well as lactoferrin and immunoglobulin A are partially digested in the preterm stomach.

Conclusions: Most human milk proteases are active in milk but not at preterm infant gastric pH. Only cathepsin D and plasmin have potential to continue degrading milk proteins within the preterm infant stomach.

FIGURE 1

Protease inhibitors in mother’s milk and in the preterm infant stomach (24–32 wk GA, 2–47 days of postnatal age). Concentration of (A) α₁-antitrypsin, (B) α₁-antichymotrypsin, (C) antithrombin III, (D) α₂-antiplasmin and (E) plasma serine protease inhibitor. (F) Peptide counts for α₁-antitrypsin. Values are mean ± SEM. Asterisks represent the p-value (*** = P < 0.001; ** = P < 0.01) for N = 18 using the paired t-tests between milk (white bar) and gastric (grey bar) samples.

FIGURE 2

Concentration of (A) lactoferrin, (B) IgA and (E) TGF-β1 in mother’s milk and in the preterm infant stomach (24–32 wk GA, 2–47 days of postnatal age). Peptide counts of (A) lactoferrin and (B) IgA in mother’s milk and in the preterm infant stomach (24–32 wk GA, 2–47 days of postnatal age. Values are mean ± SEM. Asterisks represent the p-value (*** = P < 0.001; ** = P < 0.01; * = P < 0.05) for N = 18 using the paired t-tests between milk (white bar) and gastric (grey bar) samples.

FIGURE 3

Concentrations of active (A) cathepsin D, (B) elastase and (C) cytosol aminopeptidase in mother’s milk and in the preterm infant stomach (24–32 wk GA, 2–47 days of postnatal age). Values are mean ± SEM. Asterisks represent the p-value (*** = P < 0.001) for N = 18 using the paired t-tests between milk (white bar) and gastric (grey bar) samples.