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Breastmilk-Saliva Interactions Boost Innate Immunity by Regulating the Oral Microbiome in Early Infancy.

Al-Shehri SS, Knox CL, Liley HG, Cowley DM, Wright JR, Henman MG, Hewavitharana AK, Charles BG, Shaw PN, Sweeney EL, Duley JA - PLoS ONE (2015)

Bottom Line: During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors.Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral-and hence gut-microbiota.Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, The University of Queensland, Brisbane, Australia; College of Applied Medical Science, Taif University, Taif, Saudi Arabia.

ABSTRACT

Introduction: Xanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined.

Results: Median concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3 ± 12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2-449) compared to adults (620 U/L, 48-1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns.

Discussion and conclusion: During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral-and hence gut-microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

No MeSH data available.


Related in: MedlinePlus

Breastmilk XO generates H2O2 when interacted with neonatal saliva.(A) Distribution of XO activity in breastmilk. Samples of colostrum were collected 1–5 day postpartum (n = 24). Error bars show mean±SD. XO activity was 8.0 ± 5.3 U/L. (B) Dose dependent inhibition of breastmilk XO by oxypurinol. Pooled breastmilk samples containing 0–25 μM oxypurinol concentrations (n = 3) were incubated with the peroxidase reagent containing 400 uM hypoxanthine. (C) The mean concentration of H2O2 in fresh breastmilk samples (n = 22) was 27.3±12.2 μM. (D) Kinetics of H2O2 generation after mixing 33 μL of diluted breastmilk, 33 μL of 1/3 diluted neonatal saliva and 34 μL peroxidase assay working solution (green circles). The negative control was buffer and peroxidase reagents (blue squares), (n = 2). The original concentrations of hypoxanthine and xanthine in the neonatal saliva were 70 μM and 30 μM respectively. Calculation of [H2O2] was based on two-fold dilutions assuming that breastmilk and neonatal saliva is 1:1 during suckling (1 mole xanthine produces 1 mole H2O2, 1 mole hypoxanthine generates 2 moles H2O2).
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pone.0135047.g002: Breastmilk XO generates H2O2 when interacted with neonatal saliva.(A) Distribution of XO activity in breastmilk. Samples of colostrum were collected 1–5 day postpartum (n = 24). Error bars show mean±SD. XO activity was 8.0 ± 5.3 U/L. (B) Dose dependent inhibition of breastmilk XO by oxypurinol. Pooled breastmilk samples containing 0–25 μM oxypurinol concentrations (n = 3) were incubated with the peroxidase reagent containing 400 uM hypoxanthine. (C) The mean concentration of H2O2 in fresh breastmilk samples (n = 22) was 27.3±12.2 μM. (D) Kinetics of H2O2 generation after mixing 33 μL of diluted breastmilk, 33 μL of 1/3 diluted neonatal saliva and 34 μL peroxidase assay working solution (green circles). The negative control was buffer and peroxidase reagents (blue squares), (n = 2). The original concentrations of hypoxanthine and xanthine in the neonatal saliva were 70 μM and 30 μM respectively. Calculation of [H2O2] was based on two-fold dilutions assuming that breastmilk and neonatal saliva is 1:1 during suckling (1 mole xanthine produces 1 mole H2O2, 1 mole hypoxanthine generates 2 moles H2O2).

Mentions: In view of the unexpected presence of high levels of xanthine and hypoxanthine in human neonatal saliva, we formed a hypothesis that this phenomenon must be linked to the presence of high activity XO in mammalian milk. We assayed the in situ XO activity–localised within the milk fat globule membranes–of 24 breastmilk samples found a mean of 8.0±5.3 U/L (apparent Km = 12.8±2.1 μM, and apparent Vmax = 8.9±6.2 μmol/min) (Fig 2A). We could not detect any XO activity in pasteurised breastmilk, and not in infant dried-milk formula nor in pasteurised bovine milk, demonstrating that milk XO is inactive by commercial heat-pasteurisation. H2O2 production by breastmilk was inhibited by oxypurinol (an XO-specific inhibitor) (Fig 2B) in a dose-dependent manner, complete inhibition being achieved with 12 μM oxypurinol (apparent Ki = 0.6 μM), confirming that the role of XO activity in H2O2 generation.


Breastmilk-Saliva Interactions Boost Innate Immunity by Regulating the Oral Microbiome in Early Infancy.

Al-Shehri SS, Knox CL, Liley HG, Cowley DM, Wright JR, Henman MG, Hewavitharana AK, Charles BG, Shaw PN, Sweeney EL, Duley JA - PLoS ONE (2015)

Breastmilk XO generates H2O2 when interacted with neonatal saliva.(A) Distribution of XO activity in breastmilk. Samples of colostrum were collected 1–5 day postpartum (n = 24). Error bars show mean±SD. XO activity was 8.0 ± 5.3 U/L. (B) Dose dependent inhibition of breastmilk XO by oxypurinol. Pooled breastmilk samples containing 0–25 μM oxypurinol concentrations (n = 3) were incubated with the peroxidase reagent containing 400 uM hypoxanthine. (C) The mean concentration of H2O2 in fresh breastmilk samples (n = 22) was 27.3±12.2 μM. (D) Kinetics of H2O2 generation after mixing 33 μL of diluted breastmilk, 33 μL of 1/3 diluted neonatal saliva and 34 μL peroxidase assay working solution (green circles). The negative control was buffer and peroxidase reagents (blue squares), (n = 2). The original concentrations of hypoxanthine and xanthine in the neonatal saliva were 70 μM and 30 μM respectively. Calculation of [H2O2] was based on two-fold dilutions assuming that breastmilk and neonatal saliva is 1:1 during suckling (1 mole xanthine produces 1 mole H2O2, 1 mole hypoxanthine generates 2 moles H2O2).
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pone.0135047.g002: Breastmilk XO generates H2O2 when interacted with neonatal saliva.(A) Distribution of XO activity in breastmilk. Samples of colostrum were collected 1–5 day postpartum (n = 24). Error bars show mean±SD. XO activity was 8.0 ± 5.3 U/L. (B) Dose dependent inhibition of breastmilk XO by oxypurinol. Pooled breastmilk samples containing 0–25 μM oxypurinol concentrations (n = 3) were incubated with the peroxidase reagent containing 400 uM hypoxanthine. (C) The mean concentration of H2O2 in fresh breastmilk samples (n = 22) was 27.3±12.2 μM. (D) Kinetics of H2O2 generation after mixing 33 μL of diluted breastmilk, 33 μL of 1/3 diluted neonatal saliva and 34 μL peroxidase assay working solution (green circles). The negative control was buffer and peroxidase reagents (blue squares), (n = 2). The original concentrations of hypoxanthine and xanthine in the neonatal saliva were 70 μM and 30 μM respectively. Calculation of [H2O2] was based on two-fold dilutions assuming that breastmilk and neonatal saliva is 1:1 during suckling (1 mole xanthine produces 1 mole H2O2, 1 mole hypoxanthine generates 2 moles H2O2).
Mentions: In view of the unexpected presence of high levels of xanthine and hypoxanthine in human neonatal saliva, we formed a hypothesis that this phenomenon must be linked to the presence of high activity XO in mammalian milk. We assayed the in situ XO activity–localised within the milk fat globule membranes–of 24 breastmilk samples found a mean of 8.0±5.3 U/L (apparent Km = 12.8±2.1 μM, and apparent Vmax = 8.9±6.2 μmol/min) (Fig 2A). We could not detect any XO activity in pasteurised breastmilk, and not in infant dried-milk formula nor in pasteurised bovine milk, demonstrating that milk XO is inactive by commercial heat-pasteurisation. H2O2 production by breastmilk was inhibited by oxypurinol (an XO-specific inhibitor) (Fig 2B) in a dose-dependent manner, complete inhibition being achieved with 12 μM oxypurinol (apparent Ki = 0.6 μM), confirming that the role of XO activity in H2O2 generation.

Bottom Line: During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors.Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral-and hence gut-microbiota.Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, The University of Queensland, Brisbane, Australia; College of Applied Medical Science, Taif University, Taif, Saudi Arabia.

ABSTRACT

Introduction: Xanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined.

Results: Median concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3 ± 12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2-449) compared to adults (620 U/L, 48-1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns.

Discussion and conclusion: During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral-and hence gut-microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

No MeSH data available.


Related in: MedlinePlus