Transcript Hydrogen Peroxide-Dependent Conversion of

Hydrogen Peroxide-Dependent
Conversion of Nitrite to Nitrate as an
Essential Feature of Bovine Milk Catalase
Nissim Silanikove, Agricultural
Research Organization, Institute of
Animal Science, Israel.
Gabriel Leitner, The Veterinary Institute, Israel
Scenario of NO cycling and metabolism
in mammary secretion (Free radicals Biol Med,
2005)
Xanthine dose-dependently enhance the
conversion of nitrite to nitrate.
45
40
No xanthine
Nitrite concentration, M
35
10 M xanthine
30
25
50 M xanthine
20
15
10
100 M xanthine
5
150 M xanthine
0
-5
0
10
20
30
40
50
60
Time, min
Under the experimental conditions,
approximately 40 µM of
xanthine are converted to urate via XO within 2 h
Silanikove et al, Journal of Agriculture Chemistry Food Science, 2009
50
Xa-0
Xa-10
Xa-50
Xa-100
Xa-150
M
40
30
20
10
0
Lg10 Nitrite Conc., M
0
1.8
20
40
60
80
Time, Min
1.6
1.4
1.2
1.0
rate constant min -1 x 1000
0
10
20
30
40
Time, Min
20
15
10
5
0
0
50
100
150
Xanthin concentration, M
200
Relative changes in lipid oxidation in milk
Relative changes in lipid oxidation, %
160
140
120
100
80
60
40
20
0
A
B
C
D
Treatments
milk stored for 6 hours in the dark at 4 0C (A), Effects of
catalase inhibitor (B), nitrite (10 mM) (C) and nitrite + catalase
(D) inhibitor
Silanikove et al, Journal of Agriculture Chemistry Food Science, 2009
22
No addition
+ 30 mM 3-AT
Nitrate, M
20
18
16
14
12
0
1
2
3
4
5
days
5
No addition
+ 30 mM 3-AT
Nitrite, M
4
3
2
1
0
0
1
2
3
days
4
5
Effect of
storing raw
milk in the
dark at 4 0C
with and
without
catalase
inhibitor
Silanikove et al,
Journal of
Agriculture
Chemistry Food
Science, 2009
Nitrotyrosine, nM g -1
500
No addition
+ 30 mM 3-AT
400
300
200
100
0
0
1
2
4
5
days
2000
Carbonyls, nM g -1
3
No addition
+ 30 mM 3-AT
1500
1000
500
Effect of
storing raw
milk in the
dark at 4
0C with and
without
catalase
inhibitor
Lipid peroxides, mEq g
-1
0
0
4.0
1
2
3
4
5
days
No addition
+ 30 mM 3-AT
3.5
3.0
2.5
2.0
0
1
2
3
days
4
5
Silanikove et al,
Journal of
Agriculture
Chemistry Food
Science, 2009
Conclusions Regarding the Control
of Oxidative Stability in Milk
XO and catalase works interactively as an antioxidant
system
Formation of nitrogen dioxide is a key process in oxidative
stress in milk. Thus, controlling this process should improve
milk oxidative stability
The function of catalase is rate limited by hydrogen
peroxide, which is provided by the activity of XO
Effect of LPS: 6 cows served as control, in second
set of six cows one of the front and one rear glands
were treated with LPS (10 ml with 10 µg/ ml LPS)
while the contra-lateral glands served as running
control. The cows milk were sampled at -24h, 0 h
(before treatment) and 24, 48 and 76 h posttreatment.
+
The data were analyzed
for the effect of treatment
and time at a single gland
level
+
-
Effect of LPS on milk yield
20
Milk Yield (L/day)
18
16
14
12
10
-25
0
25
50
Time in Relation to LPS Challange (h)
75
Effect of LPS on lactose concentration
5.6
A
Lactose Concentration (%)
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
-25
0
25
50
Time in Relation to LPS Challange (h)
75
Effect of LPS on whey proteins concentration
1.0
Whey Proteins Concentration (%)
B
0.9
0.8
0.7
0.6
0.5
-25
0
25
50
Time in Relation to LPS Challange (h)
75
Proteose-peptone Concentration (g/ml)
Effect of LPS on proteose peptones concentration
180
C
160
140
120
100
80
60
40
-25
0
25
50
Time in Relation to LPS Challange (h)
75
Effect of LPS lactoferrin concentration
250
Lactoferrin Concentration (µg/ml)
A
200
150
100
50
-25
0
25
50
Time in Relation to LPS Challange (h)
75
Effect of LPS on XO activity and urate concentration
Effect of LPS
on LPO activity,
nitrite and
nitrotyrosine
concentrations
Effect of LPS
on catalase
activity, and
nitrate
concentrations
Updated scenario of NO-cycling in milk
Novel findings:
Effect of LPS on
lactate, malate
and citrate
concentrations
Cytosolic and mitochondrial glycolysis
Lactic acid metabolism
Cytosolic formation of malic acid
2 Pyruvic
1
acid + CO2 + ATP Pyruvate carboxylase Oxaloacetic acid
+ ADP
2
Oxaloacetic
1
acid + NADH
+
NAD
2
Malic dehydrogenase
Malic acid +
Low lactose and high lactic acid in broth media affect
the growth of pathogenic type of E coli
CONCLUSIONS
The acute conversion of the epithelial cells metabolism
from principally mitochondrial-oxidative to principally
cytosolic (glycolysis) allows the diversion of metabolic
resources normally used to synthesize milk to support
the immune system.
In turn, the acute increase in the concentration of lactate
and malate in milk and the parallel reduction in lactose
concentration are probably effective mean in restraining
invading E Coli growth.
SPECULTIVE CONCLUSION
The main function of PMN
under acute inflammation is to
backup for the disruption of
the epithelial tight junction
integrity in order to prevent
sepsis and lethality