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Hydrogen Peroxide-Dependent Conversion of Nitrite to Nitrate as an
Essential Feature of Bovine Milk Catalase
Nissim Silanikove, Agricultural , Agricultural Research Organization, Institute of Research Organization, Institute of
Animal Science, Israel.Animal Science, Israel.
Gabriel Leitner, The Veterinary Institute, Israel, The Veterinary Institute, Israel
Scenario of NO cycling and metabolism in mammary secretion (Free radicals Biol Med,
2005)
Xanthine dose-dependently enhance the Xanthine dose-dependently enhance the
conversion of nitrite to nitrateconversion of nitrite to nitrate . .
0 10 20 30 40 50 60-5
0
5
10
15
20
25
30
35
40
45
150 M xanthine
100 M xanthine
50 M xanthine
10 M xanthine
No xanthine
Nitri
te c
on
ce
ntr
atio
n, M
Time, min
Silanikove et al, Journal of Agriculture Chemistry Food Science, 2009
Under the experimental conditions, approximately 40 µM of Under the experimental conditions, approximately 40 µM of xanthine are converted to urate via XO within 2 hxanthine are converted to urate via XO within 2 h
0 20 40 60 800
10
20
30
40
50Xa-0
Xa-10Xa-50Xa-100Xa-150
Time, Min M
0 10 20 30 401.0
1.2
1.4
1.6
1.8
Time, Min
Lg 1
0 N
itri
te C
on
c.,
M
0 50 100 150 2000
5
10
15
20
Xanthin concentration, M
rate
co
nsta
nt
min
-1 x
1000
Relative changes in lipid oxidation in milk Relative changes in lipid oxidation in milk
A B C D0
20
40
60
80
100
120
140
160
Re
lativ
e c
ha
nge
s in
lipi
d o
xid
atio
n, %
Treatments
Silanikove et al, Journal of Agriculture Chemistry Food Science, 2009
milk stored for 6 hours in the dark at 4 0milk stored for 6 hours in the dark at 4 0CC (A), Effects of (A), Effects of catalase inhibitor (B), nitrite (10 mM) (C) and nitrite + catalase catalase inhibitor (B), nitrite (10 mM) (C) and nitrite + catalase (D) inhibitor(D) inhibitor
0 1 2 3 4 512
14
16
18
20
22No addition+ 30 mM 3-AT
days
Nitr
ate,
M
0 1 2 3 4 50
1
2
3
4
5No addition+ 30 mM 3-AT
days
Nitr
ite,
MEffect of Effect of storing raw storing raw milk in the milk in the darkdark at 4 0 at 4 0C C
with and with and without without catalase catalase
inhibitorinhibitor Silanikove et al, Journal of Agriculture Chemistry Food Science, 2009
0 1 2 3 4 50
100
200
300
400
500No addition+ 30 mM 3-AT
days
Nit
roty
rosin
e, n
M g-1
0 1 2 3 4 50
500
1000
1500
2000No addition+ 30 mM 3-AT
days
Car
bo
nyl
s, n
M g
-1
0 1 2 3 4 52.0
2.5
3.0
3.5
4.0No addition+ 30 mM 3-AT
days
Lip
id p
ero
xid
es,
mE
q g
-1
Effect of Effect of storing raw storing raw milk in the milk in the
dark at 4 dark at 4 00C C with and with and
without without catalase catalase inhibitorinhibitor
Silanikove et al, Journal of Agriculture Chemistry Food Science, 2009
Conclusions Regarding the Control Conclusions Regarding the Control of Oxidative Stability in Milkof 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 post-treatment.
+
+ -
The data were analyzed for the effect of treatment and time at a single gland
level
-25 0 25 50 7510
12
14
16
18
20
Milk
Yie
ld (
L/da
y)
Time in Relation to LPS Challange (h)
Effect of LPS on milk yield
-25 0 25 50 754.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6A
Lact
ose
Con
cent
ratio
n (%
)
Time in Relation to LPS Challange (h)
Effect of LPS on lactose concentration
-25 0 25 50 75
0.5
0.6
0.7
0.8
0.9
1.0B
Whey P
rote
ins C
oncentr
ation (
%)
Time in Relation to LPS Challange (h)
Effect of LPS on whey proteins concentration
-25 0 25 50 75
40
60
80
100
120
140
160
180C
Pro
teo
se-p
epto
ne
Conce
ntr
ation
(g
/ml)
Time in Relation to LPS Challange (h)
Effect of LPS on proteose peptones concentration
-25 0 25 50 7550
100
150
200
250A
La
cto
ferr
in C
on
ce
ntr
atio
n (
µg
/ml)
Time in Relation to LPS Challange (h)
Effect of LPS lactoferrin concentration
Effect of LPS on XO activity and urate concentration
Effect of LPS on LPO activity, nitrite and nitrotyrosineconcentrations
Effect of LPS on catalase activity, and nitrateconcentrations
Updated scenario of NO-cycling in milk
Novel findings: Effect of LPS on lactate, malate and citrateconcentrations
Cytosolic and mitochondrial glycolysis
Lactic acid metabolism
Cytosolic formation of malic acid
2 Pyruvic acid + CO2 + ATP Pyruvate carboxylase Oxaloacetic acid 1
+ ADP 2
Oxaloacetic acid + NADH Malic dehydrogenase Malic acid + 1
NAD+ 2
Low lactose and high lactic acid in broth media affect the growth of pathogenic type of E coli
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.
CONCLUSIONS
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
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