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Antibacterial properties of xanthine oxidase in human milk
RESEARCH LETTERS
Antibacterial properties of xanthine oxidase in human milk C R Stevens, T M Millar, J G Clinch, J M Kanczler, T Bodamyali, D R Blake Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.
It is widely accepted that formula-fed babies show increased incidence of infective gastroenteritis. The incidence is greater in less developed countries.1 While breastfeeding continues to be widely promoted in these countries there is growing concern regarding the vertical transmission of HIV-1 via human milk, creating a dilemma for HIV-1 infected mothers.1,2 Here we report the discovery of natural antibacterial properties of xanthine oxidase in mammalian milk, and that these properties are lacking in formula-milk preparations. Xanthine oxidase, a cellular redox enzyme, is highly expressed in mammary epithelial cells. During lactation these cells synthesise milk-fat globules that are packaged in a membrane of which xanthine oxidase is the predominant protein, thus milk is a rich source of the enzyme. Xanthine oxidase can generate weakly microbicidal superoxide and hydrogen peroxide. These may give antimicrobial protection to the neonatal stomach. However, superoxide and hydrogen peroxide production are greatest at a pH above 7 and require the presence of oxygen; the pH of the neonatal stomach generally ranges between pH 4 and pH 6,3 with an oxygen concentration at or below 5%. The neonatal stomach environment provides the right conditions for xanthine
oxidase to generate nitric oxide from organic and inorganic nitrates and nitrites, which we have already described.4 These findings, in combination with the hitherto unaccountably high concentrations of xanthine oxidase in mammalian milk, led us to propose an antibiotic role for xanthine-oxidase-derived nitric oxide and its derivatives in neonatal nutrition. In support of this, nitric oxide itself is a well-known microbicidal species that, in combination with superoxide, generates the potent microbicide, peroxynitrite.5 Nitric oxide is generated in human milk via the action of endogenous xanthine oxidase on nitrite with xanthine, hypoxanthine, and reduced nicotinamide adenine nucleotide as electron-donating substrates. Series of milk samples from 25 fullterm mothers were collected prospectively between days 2 and 112 post partum. 175 samples were analysed for the ability to generate nitric oxide with a Sievers NOA 280 (Analytix Limited, Peterlee, UK), which detects chemiluminescence of gaseous phase nitric oxide after reaction with ozone. Standard assay conditions were oxygen concentration below 5%, pH 5·5, 37°C, sodium nitrite (10 mmol/L), reduced nicotinamide adenine dinucleotide or hypoxanthine (100 mol/L). Nitric oxide production was followed until steady state was attained (about 20 mins). The addition of oxypurinol (a specific xanthine oxidase inhibitor) at this time stopped the generation of nitric oxide, confirming xanthine oxidase to be critical in this reaction. All milk samples showed nitric-oxide-generating capacity of between 52 and 465 nmol/min per mL (greatest activity seen in hind-milk samples). Activity correlated with time post partum (figure 1); greatest nitric-oxide-generating activity was measured during the first 3 weeks (mean xanthine oxidase protein concentration was 510 g/mL) but remained greater than 110 g/mL for 112 days. Activity was not detected in any formula preparation tested; these included SMA White, SMA Gold, Aptamil, Cow and Gate Premium, Nestlé Lactogen, and Farley’s First Milk. The time of peak xanthine oxidase activity correlates with that of greatest susceptibility of the neonate to enteric pathogens and with a study showing breastmilk affords the greatest protection in infants less than 2 months of age (pooled odds ratio: 5·8 [95% CI 3·4–9·8]).1 Hypothesising that the
Mean nitric-oxide generation (nmol/min per mL)
250
200
150
100
50
0 1-5
6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 75-80 Days post partum
Figure 1: Mean nitric-oxide generation, in 5 day ranges post partum, in breastmilk samples from 25 mothers
THE LANCET • Vol 356 • September 2, 2000
829
For personal use only. Not to be reproduced without permission of The Lancet.
RESEARCH LETTERS
a complete abrogation of bacterial overgrowth in milk over 7 days (figure 2, B). Since the natural antibacterial properties of xanthine oxidase in breastmilk are absent in current formula preparations, we propose that better retention of the enzyme activity will increase the safety of breastmilk substitutes for use in situations where the high risk of vertical transmission of HIV-1 renders breastfeeding undesirable. Ways of achieving the latter have been patented accordingly, International Patent PCT/GB99/02845—ingestible compositions comprising antibacterial agents 28/08/99.
A 120
Viable control (%)
100
80
60
We thank the University of Bath, South and West Regional NHS, and the Royal National Hospital for the Rheumatic Diseases, Bath, for financial support. We thank all the mothers who provided milk samples.
40
1
Hydrogen peroxide 20
2
Hypoxanthine
3
0 1
10
100
1000
(mol/L)
4
B 5
100 000
Department of Medical Sciences, University of Bath, Bath (C R Stevens PhD, T M Millar PhD, J G Clinch MRCP, J M Kanczler PhD, T Bodamyali PhD, D R Blake FRCP) and Royal National Hospital for the Rheumatic Diseases, Bath, UK (C R Stevens, J G Clinch, D R Blake)
Colony forming units per mL
10 000
Correspondence to: Dr Cliff R Stevens, Department of Medical Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK (e-mail: [email protected])
1000
100
10
Somatic mutation in MECP2 as a non-fatal neurodevelopmental disorder in males
Control Hypoxanthine
J Clayton-Smith, P Watson, S Ramsden, G C M Black
0 0
4 5 6 7 Time (days) Figure 2: A: inhibitory effect of xanthine oxidase in the presence of hypoxanthine and hydrogen peroxide on the growth rate of E coli (also representative of Salmonella); B: effect of hypoxanthine (100 mol/L) addition to milk on bacterial growth at room temperature
1
2
3
8
Results are mean (SE) in triplicate (n=3).
xanthine oxidase content of breastmilk contributes to its protective nature, we studied the effect of xanthine oxidase and hypoxanthine-derived species on the growth rates of the common enterobacteriacae Escherichia coli and Salmonella enteritidis and compared this with the effect of the antibacterial agent hydrogen peroxide. We also examined the antibacterial effect of enhancing endogenous xanthine oxidase activity in fresh milk by addition of hypoxanthine (100 mol/L). Xanthine oxidase, even at a low concentration (150 g/mL) in the range of endogenous milk enzyme (110–680 g/mL), showed potent growth-inhibitory activity. This was dosedependent on hypoxanthine concentration, the effect being greater than that of hydrogen peroxide for both E coli and S enteritidis (figure 2, A). The addition of hypoxanthine, increasing endogenous milk xanthine oxidase activity, caused
830
Victora CG. Effect of breastfeeding on infant and child mortality due to infectious diseases in less developed countries: a pooled analysis. Lancet 2000; 355: 451–55. Nduati R, John G, Mbori-Ngacha D, et al. Effect of breastfeeding and formula feeding on transmission of HIV-1: a randomized clinical trial. JAMA 2000; 283: 1167–74. Widstrom AM, Christensson K, Ransjo-Arvidson AB, Matthiesen AS, Wimberg J, Uvnas-Moberg K. Gastric aspirates of newborn infants: pH, volume and levels of gastrin and somatostatin-like immunoreactivity. Acta Pediatr Scand 1988; 77: 502–08. Millar TM, Stevens CR, Benjamin N, Eisenthal R, Harrison R, Blake DR. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Letts 1998; 427: 225–28. Brunelli L, Crow JP, Beckman JS. The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys 1995; 316: 327–34.
Rett syndrome is a cause of severe learning disability in girls and is associated with a characteristic history and movement disorder. It is an X-linked dominant condition associated with mutations of the MECP2 gene on the distal part of the X-chromosome. If present in a male conceptus, the mutation is usually lethal. We present evidence to show that males can be affected by Rett syndrome. In the boy presented, this situation came about because cells containing the MECP2 mutation existed alongside a normal cell line. Somatic mosaicism could explain the occurrence of other X-linked dominant disorders in males, when they would normally be lethal.
Rett syndrome is a childhood neurodevelopmental disorder almost exclusively affecting girls1 who develop normally for the first few months of life before undergoing a period of regression with loss of purposeful hand use and speech. Patients develop stereotypic hand-wringing movements, with ataxia and episodes of hyperventilation. The disorder stabilises in later childhood but severe mental retardation generally remains. A gene for Rett syndrome (MECP2) has been identified on the X chromosome2 that binds to single methylated CpG base pairs (where a cytosine residue lies next to a guanine residue in the DNA sequence) throughout the genome and “silences” other genes.3 Rett syndrome is thought to arise because of excessive transcriptional noise due to failure of gene silencing by MECP2.
THE LANCET • Vol 356 • September 2, 2000
For personal use only. Not to be reproduced without permission of The Lancet.
Antibacterial properties of xanthine oxidase in human milk C R Stevens, T M Millar, J G Clinch, J M Kanczler, T Bodamyali, D R Blake Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.
It is widely accepted that formula-fed babies show increased incidence of infective gastroenteritis. The incidence is greater in less developed countries.1 While breastfeeding continues to be widely promoted in these countries there is growing concern regarding the vertical transmission of HIV-1 via human milk, creating a dilemma for HIV-1 infected mothers.1,2 Here we report the discovery of natural antibacterial properties of xanthine oxidase in mammalian milk, and that these properties are lacking in formula-milk preparations. Xanthine oxidase, a cellular redox enzyme, is highly expressed in mammary epithelial cells. During lactation these cells synthesise milk-fat globules that are packaged in a membrane of which xanthine oxidase is the predominant protein, thus milk is a rich source of the enzyme. Xanthine oxidase can generate weakly microbicidal superoxide and hydrogen peroxide. These may give antimicrobial protection to the neonatal stomach. However, superoxide and hydrogen peroxide production are greatest at a pH above 7 and require the presence of oxygen; the pH of the neonatal stomach generally ranges between pH 4 and pH 6,3 with an oxygen concentration at or below 5%. The neonatal stomach environment provides the right conditions for xanthine
oxidase to generate nitric oxide from organic and inorganic nitrates and nitrites, which we have already described.4 These findings, in combination with the hitherto unaccountably high concentrations of xanthine oxidase in mammalian milk, led us to propose an antibiotic role for xanthine-oxidase-derived nitric oxide and its derivatives in neonatal nutrition. In support of this, nitric oxide itself is a well-known microbicidal species that, in combination with superoxide, generates the potent microbicide, peroxynitrite.5 Nitric oxide is generated in human milk via the action of endogenous xanthine oxidase on nitrite with xanthine, hypoxanthine, and reduced nicotinamide adenine nucleotide as electron-donating substrates. Series of milk samples from 25 fullterm mothers were collected prospectively between days 2 and 112 post partum. 175 samples were analysed for the ability to generate nitric oxide with a Sievers NOA 280 (Analytix Limited, Peterlee, UK), which detects chemiluminescence of gaseous phase nitric oxide after reaction with ozone. Standard assay conditions were oxygen concentration below 5%, pH 5·5, 37°C, sodium nitrite (10 mmol/L), reduced nicotinamide adenine dinucleotide or hypoxanthine (100 mol/L). Nitric oxide production was followed until steady state was attained (about 20 mins). The addition of oxypurinol (a specific xanthine oxidase inhibitor) at this time stopped the generation of nitric oxide, confirming xanthine oxidase to be critical in this reaction. All milk samples showed nitric-oxide-generating capacity of between 52 and 465 nmol/min per mL (greatest activity seen in hind-milk samples). Activity correlated with time post partum (figure 1); greatest nitric-oxide-generating activity was measured during the first 3 weeks (mean xanthine oxidase protein concentration was 510 g/mL) but remained greater than 110 g/mL for 112 days. Activity was not detected in any formula preparation tested; these included SMA White, SMA Gold, Aptamil, Cow and Gate Premium, Nestlé Lactogen, and Farley’s First Milk. The time of peak xanthine oxidase activity correlates with that of greatest susceptibility of the neonate to enteric pathogens and with a study showing breastmilk affords the greatest protection in infants less than 2 months of age (pooled odds ratio: 5·8 [95% CI 3·4–9·8]).1 Hypothesising that the
Mean nitric-oxide generation (nmol/min per mL)
250
200
150
100
50
0 1-5
6-10 11-15 16-20 21-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 66-70 71-75 75-80 Days post partum
Figure 1: Mean nitric-oxide generation, in 5 day ranges post partum, in breastmilk samples from 25 mothers
THE LANCET • Vol 356 • September 2, 2000
829
For personal use only. Not to be reproduced without permission of The Lancet.
RESEARCH LETTERS
a complete abrogation of bacterial overgrowth in milk over 7 days (figure 2, B). Since the natural antibacterial properties of xanthine oxidase in breastmilk are absent in current formula preparations, we propose that better retention of the enzyme activity will increase the safety of breastmilk substitutes for use in situations where the high risk of vertical transmission of HIV-1 renders breastfeeding undesirable. Ways of achieving the latter have been patented accordingly, International Patent PCT/GB99/02845—ingestible compositions comprising antibacterial agents 28/08/99.
A 120
Viable control (%)
100
80
60
We thank the University of Bath, South and West Regional NHS, and the Royal National Hospital for the Rheumatic Diseases, Bath, for financial support. We thank all the mothers who provided milk samples.
40
1
Hydrogen peroxide 20
2
Hypoxanthine
3
0 1
10
100
1000
(mol/L)
4
B 5
100 000
Department of Medical Sciences, University of Bath, Bath (C R Stevens PhD, T M Millar PhD, J G Clinch MRCP, J M Kanczler PhD, T Bodamyali PhD, D R Blake FRCP) and Royal National Hospital for the Rheumatic Diseases, Bath, UK (C R Stevens, J G Clinch, D R Blake)
Colony forming units per mL
10 000
Correspondence to: Dr Cliff R Stevens, Department of Medical Sciences, University of Bath, Claverton Down, Bath BA2 7AY, UK (e-mail: [email protected])
1000
100
10
Somatic mutation in MECP2 as a non-fatal neurodevelopmental disorder in males
Control Hypoxanthine
J Clayton-Smith, P Watson, S Ramsden, G C M Black
0 0
4 5 6 7 Time (days) Figure 2: A: inhibitory effect of xanthine oxidase in the presence of hypoxanthine and hydrogen peroxide on the growth rate of E coli (also representative of Salmonella); B: effect of hypoxanthine (100 mol/L) addition to milk on bacterial growth at room temperature
1
2
3
8
Results are mean (SE) in triplicate (n=3).
xanthine oxidase content of breastmilk contributes to its protective nature, we studied the effect of xanthine oxidase and hypoxanthine-derived species on the growth rates of the common enterobacteriacae Escherichia coli and Salmonella enteritidis and compared this with the effect of the antibacterial agent hydrogen peroxide. We also examined the antibacterial effect of enhancing endogenous xanthine oxidase activity in fresh milk by addition of hypoxanthine (100 mol/L). Xanthine oxidase, even at a low concentration (150 g/mL) in the range of endogenous milk enzyme (110–680 g/mL), showed potent growth-inhibitory activity. This was dosedependent on hypoxanthine concentration, the effect being greater than that of hydrogen peroxide for both E coli and S enteritidis (figure 2, A). The addition of hypoxanthine, increasing endogenous milk xanthine oxidase activity, caused
830
Victora CG. Effect of breastfeeding on infant and child mortality due to infectious diseases in less developed countries: a pooled analysis. Lancet 2000; 355: 451–55. Nduati R, John G, Mbori-Ngacha D, et al. Effect of breastfeeding and formula feeding on transmission of HIV-1: a randomized clinical trial. JAMA 2000; 283: 1167–74. Widstrom AM, Christensson K, Ransjo-Arvidson AB, Matthiesen AS, Wimberg J, Uvnas-Moberg K. Gastric aspirates of newborn infants: pH, volume and levels of gastrin and somatostatin-like immunoreactivity. Acta Pediatr Scand 1988; 77: 502–08. Millar TM, Stevens CR, Benjamin N, Eisenthal R, Harrison R, Blake DR. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Letts 1998; 427: 225–28. Brunelli L, Crow JP, Beckman JS. The comparative toxicity of nitric oxide and peroxynitrite to Escherichia coli. Arch Biochem Biophys 1995; 316: 327–34.
Rett syndrome is a cause of severe learning disability in girls and is associated with a characteristic history and movement disorder. It is an X-linked dominant condition associated with mutations of the MECP2 gene on the distal part of the X-chromosome. If present in a male conceptus, the mutation is usually lethal. We present evidence to show that males can be affected by Rett syndrome. In the boy presented, this situation came about because cells containing the MECP2 mutation existed alongside a normal cell line. Somatic mosaicism could explain the occurrence of other X-linked dominant disorders in males, when they would normally be lethal.
Rett syndrome is a childhood neurodevelopmental disorder almost exclusively affecting girls1 who develop normally for the first few months of life before undergoing a period of regression with loss of purposeful hand use and speech. Patients develop stereotypic hand-wringing movements, with ataxia and episodes of hyperventilation. The disorder stabilises in later childhood but severe mental retardation generally remains. A gene for Rett syndrome (MECP2) has been identified on the X chromosome2 that binds to single methylated CpG base pairs (where a cytosine residue lies next to a guanine residue in the DNA sequence) throughout the genome and “silences” other genes.3 Rett syndrome is thought to arise because of excessive transcriptional noise due to failure of gene silencing by MECP2.
THE LANCET • Vol 356 • September 2, 2000
For personal use only. Not to be reproduced without permission of The Lancet.