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