Breastmilk - saliva interaction and salivary purines
Dr John Duley School of Pharmacy, UQ Review: Functions of Nucleotides
• Chemical energy e.g. ATP • Synthesise nucleic acids (RNA/DNA) e.g. ‘A’, ‘G’ = bases Adenine and Guanine • Coenzymes e.g. NAD, FAD • Synthesis e.g. UDP-glucoseglycogen, GTPdopamine • Cell messengers e.g. cyclic-AMP, G-proteins. Review: What are nucleotides?
‘P/P Base’ + Sugar (ribose or deoxy-ribose) + Phosphates Purines (Adenine + ribose = Adenosine + 3 phosphates)
------Phosphates------Nucleoside------Sugar---
Pyrimidines For obscure reasons, we looked at SALIVA concentrations of nucleobases and nucleosides, using HPLC-MS/MS
Hypoxanthine and Xanthine in blue Orange line= median (µM) 120 Adults 120 Neonates 120 6-week Infants 100 100 100 M) (n=77) (n=60) (n=20) M) M) 80 80 80
60 60 60
40 40 40 Concentration ( Concentration Concentration ( Concentration ( Concentration 20 20 20
0 0 0
Uracil Uridine Adenine Inosine Uracil Uracil Uridine Xanthine Thymine Inosine Uridine Adenine Inosine Adenosine Guanosine XanthineAdenine Thymine Xanthine Thymine Dihydrouracil Adenosine Guanosine Adenosine Guanosine PseudouridineHypoxanthine Dihydrouracil Dihydrouracil Dihydrothymine PseudouridineHypoxanthine PseudouridineHypoxanthine Dihydrothymine Dihydrothymine Succinyladenosine Succinyladenosine Succinyladenosine
Time lines: So… We found much higher concentrations nucleosides and bases in neonatal saliva than in adult saliva (we have also found nucleotides, e.g. ATP!)
But what is the role of these metabolites in baby saliva? Theory A- To feed the gut endothelial cells? Theory B- To regulate oral microflora? Theory A : ‘To feed the gut endothelium’? Nucleotides aid gut maturation/immunity
Maybe saliva nucleotides also feed gut cells? (important for intubated premmies?) Theory B: ‘To regulate the oral microflora’?
Hypoxanthine and Xanthine in saliva These are substrates for enzyme ‘Xanthine Oxidase’ (XO)
XO has limited tissue distribution Mainly in liver and small bowel, none in saliva, but… very active in breast milk! (part of the ‘lactoperoxidase system’ that generates free radicals) Secretion of Xanthine Oxidase (XO) in Breast Milk Milk Fat Globule
Milk Fat Globule secreted in membrane
Cytoplasmic lipid droplets
Micro-lipid droplets
Rough endoplasmic Xanthine oxidase reticulum of milk inside milk fat secretory cell globule BREAST MILK: XO Activity
XO activity in 24 breast milk samples uric acid 20 40 H2O2 15 XO 30 M)
( 10 2 20 O 2 xanthine H + allopurinol 5 10
H O /L) (U oxidase Xanthine 2 2 XO 0 0 0 10 20 30 40 50 60 70 hypoxanthine Time (min)
Xanthine/Hypoxanthine + breast milk H2O2
But… does this mean there is hydrogen peroxide in breast milk? Yes! Peroxide in fresh breast milk samples
22 breast milk samples, measured in duplicate:
Hydrogen peroxide in breast milk = 28.2±12.4 µM
But OMG! What happens when baby saliva (containing lots of hypoxanthine and xanthine) mixes with breast milk? FINALLY! Breast Milk Meets Baby Saliva
0.15 Peroxide production by mixing breast milk and baby saliva 0.10
0.05 Peroxide (Absorb) Peroxide +allopurinol
0.00 0 20 40 60 80 100 Time(min)
Baby saliva How strong is peroxide in breast milk + saliva?
Milk peroxide: The maths
H2O2 Typical breast milk = 30 µM Typical baby saliva Hypoxanthine: 30 µM 60 µM Xanthine: 20 µM 20 µM 80 µM
So peroxide in baby’s mouth ~ 50-100 µM (< 1/10,000 of 3%) = ‘homeopathic peroxide’
6 : too dilute to be ‘bactericidal’? 3% H2O2 ≈ 1 mol/L = 10 µM Microbial studies: methods
Infant oral Bacteria Bacteria microbiota: Breast milk Breastfed vs Bottle 0-400µM H2O2 in ‘Oxoid’ media +”Baby saliva” Con PP +XH Allo Bacterial growth in ‘Oxoid’ titrated against H2O2
S. aureus (Gram+) Salmonella spp (Gram-) 0.5 0.5
0.4 0.4
0.3 0.3
0.2 0.2 OD (600nm) OD OD (600nm) OD 0.1 0.1
0.0 0.0 0 10 20 30 40 50 60 200 400 0 10 20 30 40 50 60 200 400 H O (M) H2O2 (M) 2 2
0.8 L. plantarum (Gram+ rod) 0.5 E. coli (Gram-)
0.4 0.6 0.3 0.4 0.2 OD (600nm) OD OD (600nm) OD 0.2 0.1
0.0 0.0 0 10 20 30 40 50 60 200 400 0 10 20 30 40 50 60 200 400 H2O2 (M) H2O2 (M) Bacterial growth in breast milk + “baby saliva” titrated against xanthine/hypoxanthine
S. aureus Salmonella spp 8 8
6 6
** 4 4 Log CFU/mL Log Log CFU/mL Log
2 2
0 6 25 50 0 6 25 50 Xanthine/hypoxanthine conc (M) Xanthine/hypoxanthine conc (M)
L. plantarum E. coli
8 8
6 6 **
4 4 Log CFU/mL Log Log CFU/mL Log
2 2
0 6 25 50 0 6 25 50 Xanthine/hypoxanthine conc (M) Xanthine/hypoxanthine conc (M) Overlay: Bacterial growth vs H2O2 vs breast milk + saliva xanthine/hypoxanthine
Salmonella spp S. aureus (Opportunistic Pathogen) (Opportunistic Pathogen) 8 0.5 0.5 8 0.4 6 0.4 0.3 6 0.3 4 0.2 OD (600nm) OD ** CFU/mL Log 4 0.2 0.1
OD (600nm) OD 2 Log CFU/mL Log 0.1 2 0.0 0 10 206 30 402550 60 50200 400 0.0 Xanthine/hypoxanthineH2O2 (M) conc ( M) 00 10 206 30 40 2550 60 50200 400 Xanthine/hypoxanthineH2O2 (M) conc (M) E. coli (Commensal) 0.8 L. plantarum (Commensal) 0.5 8 8
0.6 0.4 6 6 ** 0.3 0.4 4 40.2 OD (600nm) OD Log CFU/mL Log OD (600nm) OD 0.2 CFU/mL Log 0.1 2 2 0.0 0.0 0 10 206 30 4025 50 60 50200 400 00 10 206 30 402550 60 50200 400 Xanthine/hypoxanthineH2O2 ( M) conc ( M) Xanthine/hypoxanthineH2O2 (M) conc (M) Bactericidal effect: breast milk + saliva (PP = Purine+Pyrim mix; XH = 20uM Xan+27uM Hypoxan +/- Allopurinol)
S. aureus Salmonella spp ns 8 ** 8 ** *
* 6 6
4 4 Log CFU/mL Log Log CFU/mL Log 2 2
0 0 Con-PP PP +XH+PP Allopurinol Con-PP PP +XH+PP Allo +PP (-XH) +PP (Oxyp) +PP (-XH) +PP (Oxyp) L. plantarum E. coli 8 ns ns 8
6 6
4 4 Log CFU/mL Log
2 CFU/mL Log 2
0 0
-PP +PP Con PP +XH Allo Con-PP PP +XH+PP Allo +PP (-XH) +PP (Oxyp) +PP (-XH) +PP (Oxyp)
*p < 0.05 ** p < 0.01 *** p < 0.001 Bactericidal effect of saliva + breast milk
S. epidermidis - Streptococcus - S. pyogenes 7 6 6 5
5 4
4 3 3 2 Log CFU/mL Log
Log CFU/mL Log 2 1 1 0 0
Con-PP PP +XH+PP AlloOXY Con-PP PP +XH+PP AlloOXY +PP-HX +PP-HX
K. pneumoniae - Klebsiella Pneumococcus - P. aeruginosa 8 6 7 5 6 5 4
4 3 3
Log CFU/mL Log 2
2 CFU/mL Log 1 1 0 0
Con-PP PP +XH+PP AlloOXY Con PP +XH Allo -PP +PP OXY +PP-HX +PP-HX Bactericidal effect of saliva + breast milk
E. faecalis (VRE - VanB) E. faecium (VRE- VanA) 5 5
4 4
3 3
2 2 Log CFU/mL Log Log CFU/mL Log 1 1
0 0
-PP +PP Con-PP PP +XH+PP AlloOXY Con PP +XH OXYAllo +PP-HX +PP-HX
E. faecalis (non-VRE) Candida C. albicans 6 7 5 6
4 5 4 3 3 2 Log CFU/mL Log Log CFU/mL Log 2 1 1 0 0
-PP Con-PP PP +XH+PP AlloOXY Con PP +XH+PP OXYAllo +PP-HX +PP-HX Concluding remarks • Neonate saliva has grossly raised nucleotides, nucleosides and bases • Role of these metabolites in neonate saliva: 1. Swallowed – to feed gut endothelium? 2. In the mouth: to select commensal bacteria? • Breast milk + neonate saliva peroxide: 1. ‘Lactoperoxidase system’ ROS and RNS 2. “Innate immunity” in neonates?
Acknowledgments
• Saad Al Shehri (UQ) – for his excellent PhD • Helen Liley (Mater /UQ) – Neonatology • Christine Knox (QUT) – Microbiology • Nick Shaw (UQ) – Pharmacy • David Cowley (Mater /UQ) – Xanthine oxidase kinetics • Mike Henman (Mater) – Analytical Chemistry • NHMRC and Qld Golden Casket – for the money Baby power!
Thank you!.. ‘In vivo’ effects: breast vs bottle (pilot study) Infants: Full term Normal delivery No antibiotics (incl. mother)
Breast-fed n=26 Bottle-fed n=12
Oral swab (at 4-8 weeks old)
DNA extraction
16s rRNA gene PCR (~600k sequences)
Bioinformatics analysis Infant oral microbiota: breast vs bottle - phyla Firmacutes! (mostly Staph) 100
Unclassified;Other k__Bacteria;Other 80 k__Bacteria;p__Actinobacteria
y k__Bacteria;p__Bacteroidetes t i n
u 60 k__Bacteria;p__Cyanobacteria m
m k__Bacteria;p__Firmicutes o c
l k__Bacteria;p__Fusobacteria a t o t 40 k__Bacteria;p__Proteobacteria
% k__Bacteria;p__TM7 k__Bacteria;p__Tenericutes 20
0 Breastfed Formula fed Oral microbiota, minus Firmacutes - phyla
15 Actinobacteria Bacteroidetes Fusobacteria Proteobacteria 10
% total community total % 5
0
Breast-fed Formula-fed Oral microbiota - families
Breast-fed Formula-fed
Staphylococcaceae Gemellaceae Carnobacteriaceae Lactobacillaceae Streptococcaceae
Firmicutes Lachnospiraceae Veillonellaceae
Actinomycetaceae Micrococcaceae Formula Propionibacteriaceae less Segniliparaceae Bifidobacteriaceae diversity
Actinobacteria Coriobacteriaceae
Porphyromonadaceae
Prevotellaceae
Flavobacteriaceae Bacteroidetes Oral micribiota conclusions
Both breast-fed and formula-fed dominated by phylum Firmicutes (mostly Staph)
Higher prevalence of oral Bacteroidetes (e.g. Prevotella) in formula-fed (p=0.01)
Higher prevalence of Proteobacteria in breast-fed; not detected in formula-fed (p=0.04)
The composition of oral microbiota changed over time - e.g. prevalence of Streptococcus spp. increased 4 8 weeks, both breast-fed and formula-fed