nature publishing group Basic Science Investigation | Articles Human milk H2O2 content: does it benefit preterm infants? Monika Cieslak1, Cristina H.F. Ferreira2, Yulia Shifrin1, Jingyi Pan1 and Jaques Belik1 BACKGROUND: Human milk has a high content of the lipoxygenase, xanthine oxidase, and myeloperoxidase, con- antimicrobial compound hydrogen peroxide (H2O2). As tribute to its formation. H2O2 is bactericidal and together with opposed to healthy full-term infants, preterm neonates are milk-containing thiocyanates (9) generates, via the lactoper- fed previously expressed and stored maternal milk. These oxidase system (LPS), hypothiocyanite, a non-stable molecule practices may favor H2O2 decomposition, thus limiting its (10), that further enhances its microbicidal activity (11). potential benefit to preterm infants. The goal of this study was Human milk production is oxytocin-dependent (12). Its ’ to evaluate the factors responsible for H2O2 generation and H2O2 content is regulated by the mammary epithelial cells degradation in breastmilk. xanthine oxidase (XO) expression and activity. XO avail- METHODS: Human donors’ and rats’ milk, along with rat ability, in turn, is dependent on the expression and activity of mammary tissue were evaluated. The role of oxytocin and xanthine oxidoreductase (XOR), an enzyme that converts xanthine oxidase on H2O2 generation, its pH-dependent molybdoflavoprotein xanthine dehydrogenase (XDH) to XO stability, as well as its degradation via lactoperoxidase and via either oxidation of sulfhydryl residues, or proteolysis (13). catalase was measured in milk. XO, in the presence of the substrates hypoxanthine or RESULTS: Breast tissue xanthine oxidase is responsible for the xanthine, generates reactive oxygen species and microbicidal H2O2 generation and its milk content is dependent on molecules such as superoxide, H2O2, thiocyanate and oxytocin stimulation. Stability of the human milk H2O2 content hypothiocyanite (Figure 1). is pH-dependent and greatest in the acidic range. Complete There is limited data on the regulatory factors responsible H2O2 degradation occurs when human milk is maintained, for H2O2 generation in the mammary glands and its stability longer than 10 min, at room temperature and this process is in human milk. Addressing this knowledge gap is relevant to suppressed by lactoperoxidase and catalase inhibition. ’ our understanding of human milk sH2O2 anti-infectious CONCLUSION: Fresh breastmilk H2O2 content is labile and properties when fed to preterm infants. quickly degrades at room temperature. Further investigation As preterm infants cannot coordinate the suck–swallow on breastmilk handling techniques to preserve its H2O2 mechanism until reaching maturity, expressed human milk, content, when gavage-fed to preterm infants is warranted. or pasteurized donor milk is gavage-fed. The extent to which the maternal human milk collection, storage, and pasteuriza- tion preserves its H2O2 content has not been previously side from its superior nutritional content, when com- investigated. This issue is of clinical importance since LPS can Apared with formula, human milk provides significant induce H2O2 decomposition during milk handling and neonatal protection against infections. Breastfed infants storage process resulting in reduction or complete degrada- experience fewer and shorter bouts of infections (1) and have tion of its H2O2 content. a lower morbidity rate, when compared with formula-fed Therefore, the goal of this study was to evaluate the factors children of a similar age (2,3). Exclusive use of human milk responsible for H2O2 generation and degradation of lowers the incidence of diarrhea, urinary tract infections, and mother’s milk. otitis media in older children (4). Human milk contains an appreciable amount of hydrogen METHODS peroxide (H2O2) that peaks within days after birth, before Design declining toward the fourth postnatal week (5). H2O2 has Studies were conducted in a laboratory setting utilizing human milk been identified as a Jekyll and Hyde signaling molecule donor samples and lactating rat mammary tissue and milk, as well as ’ showing beneficial effects at physiologically relevant lower their pups milk curds. concentrations (6), but having biologically harmful properties at higher levels. Endogenously, H O is mostly generated via Animals 2 2 All procedures were conducted in accordance with the Canadian mitochondrial oxidative phosphorylation (7) and phagocytic Animals for Research Act and Canadian Council on Animal Care respiratory burst (8), but other enzymatic processes, such as regulations, and the Animal Care Committee of the Hospital 1Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute; Department of Paediatrics and Physiology, University of Toronto, Toronto, Ontario, Canada; 2Hospital das Clinicas, Ribeirão Preto, University of São Paulo, São Paulo, Brazil. Correspondence: Jaques Belik ([email protected]) Received 2 October 2017; accepted 14 November 2017; advance online publication 10 January 2018. doi:10.1038/pr.2017.303 Copyright © 2018 International Pediatric Research Foundation, Inc. Volume 83 | Number 3 | March 2018 Pediatric RESEARCH 687 Articles | Cieslak et al. Oxytocin Milk fat globule Xanthine dehydrogenase Sulfhydryl oxidase Xanthine oxidase *Hypothiocyanite Hypoxanthine Lactoperoxidase or xanthine Thiocyanate + Catalase *Superoxide *H2O2 H2O + O2 Superoxide dismutase *Bactericidal properties Figure 1. Cartoon depicting the oxytocin effect on the milk fat globule in the mammary gland and the conversion of xanthine dehydrogenase to the oxidase form. The generation of the reactive oxygen species superoxide, hydrogen peroxide (H2O2) and its conversion to hypothiocyanite by the milk-containing lactoperoxidase is also shown. approved the study protocol for Sick Children. Sprague-Dawley rats Activity Assays and H2O2 Content Measurements (Charles River, Montreal, QC, Canada) were studied. These included Tissue XD+XO and XO were assayed in the presence and absence of adult and younger animals at 1, 2, and 3 weeks of age. They were fed NAD+, respectively, as previously described (15). Briefly, rat regular rodent pellets and were housed under standard lighting and mammary glands were homogenized in 50 mM K+ phosphate buffer temperature conditions. The animals were killed by pentobarbital (pH 7.8) containing 1.0 mM EDTA, 0.2 mM PMSF, and 0.5 mM sodium injection (60 mg/kg ip (adult)), and the gastric tissue was DTT, and tissue protein content was evaluated by Bradford assay quickly excised. (Bio-Rad, Mississauga, ON, Canada). A volume of 5 μl of homo- genate was mixed with 50 μM hypoxanthine (substrate) with or + Human Milk without 1.0 mM NAD , and the increase in absorbance at 295 nm Fresh human mother’s milk samples were obtained from healthy following the formation of uric acid was used as a measure of the volunteer donors (N = 5) following the informed consent and under activity of XD+XO or XO, and normalized to protein content. One unit of tissue XD+XO, or XO was defined as the amount of enzyme a protocol approved by The Hospital for Sick Children Research μ Ethics Board. Following maternal expression, the human milk was required to form 1 mol of uric acid/min. immediately flash-frozen in liquid nitrogen, and was stored at For organ explant studies, the rat mammary glands were − 80 °C for further processing. maintained overnight in DMEM (Wisent, St-Bruno, QB, Canada) supplemented with fetal bovine serum (Wisent) and 10% antibiotics/ antimycotics (Wisent) at 37 °C, 5% CO2. Control and oxytocin- Rat Breast Tissue and Milk treated (1 μM for 1, 7, or 30 min) tissue explants were incubated in Non-lactating (control) and lactating rats (N = 3 per group) were culture medium and the H2O2 content was measured using Amplex killed, and the mammary gland tissue was quickly excised, flash- Red, as described below. − frozen in liquid nitrogen, and stored at 80 °C. Lactating rat For H O content measurement, frozen milk samples were thawed mothers’ milk was collected by a non-invasive technique reported by 2 2 and diluted 1:5 in PBS. Milk curds were homogenized in PBS. H2O2 others (14). To evaluate the oxytocin effect on the mammary gland content of mothers’ milk and pups’ milk-curd samples was enzymatic activity, lactating rats received 1.5 IU of oxytocin ip 5 min determined by the Amplex Red hydrogen peroxide/peroxidase assay prior to the tissue harvesting. kit (Molecular Probes, Invitrogen, Carlsbad, CA) according to the N = To obtain newborn gastric milk samples, 1-week-old pups ( 6) manufacturer’s protocol. Briefly, samples were incubated in the were killed by pentobarbital sodium injection 3 h after maternal reaction mixture containing 50 μM Amplex Red reagent and 0.1 U/ ’ separation. The pups stomach milk curd was retrieved and ml horseradish peroxidase (HRP), and fluorescence was measured at immediately frozen for further processing. In order to evaluate the excitation of 560 nm and emission detection at ~ 590 nm. ’ freshly ingested mother s milk, same age pups were separated for a 3- To measure the human milk LPS activity, the following procedure h period and then were returned to mothers for a 2-h period of was performed. The human milk was thawed and was maintained at breastfeeding. Pups were then killed, their gastric milk curd was room temperature for 1 h to allow complete degradation of its H O retrieved and immediately frozen for further processing. All milk and 2 2 − content. The milk was then aliquoted into