Distribution of Xanthine Dehydrogenase and Xanthine Oxidase in milk fractions :

Evidence for post-transcriptional regulation of Xanthine Oxidase in the

frame of the mammary innate immune defense system

Nissim Silanikove, Fira Shapiro, Adi Rauch & Gabriel Leitner

Or 2H2O2

NitrateNAD

Nitrite

NADH

SOD

Nitric Oxide is a Free RadicalN=O

Reaction of Lactoperoxidase with Hydrogen peroxide and

Nitrite

1 .LPO + H2O2 LPO compound 1

2. LPO compound I + NO2- LPO compound II + ●NO2

3 .LPO compound II + NO2- LPO + ●NO2

Scenario of NO cycling and metabolism in mammary secretion (Free radicals Biol Med, 2005)

Question Number 1

1. In the mammary gland, XOR has an essential, non-enzymatic, structural role in fat secretion (Vorbach et al. Genes Dev 2002, 16:3223)

2. It is well established that XOR associated with fat secretion is located within the inner side of MFGM ) e.g. J. Physiol 2002, 545:567)

Do we have sufficient XO to support its role in innate immunity? (Free radicals biol Med

2005, 38: 1139 )

Xanthine + hypoxanthine and uric acid concentration in oxytocin-induced and

mature milk

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Oxytocin Induced Mature milk

Mic

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Xanthin+HypoxanthinUric Acid

Question Number 2

As mature fresh milk do not contains measurable amount of xanthine, but contains uric acid in the range of 30 40 -micro-molar, it is important to know whether it derived from milk xanthine or secreted as uric acid?

Fresh milk (i.e., milk secreted into the alveoli within 5 to 10 min before sampling) was obtained at the end of noon-milking following injection of oxytocine.

Distribution of xanthine oxidase, alkaline phosphatase and acid phosphatase in milk fractions

Xanthine oxidase Alkaline phosphatase Acid phosphatase

% of total % of total % of total

Whoe milk 100 100 100

Fat* (MFGM) 33 45 48

WMP 21 39 34

Phos.lipids 54 84 82

Casein 3 - -

Truly soluble 43 16 18

Distribution of enzymes inside and outside of WMP membranes

XO (U/ml) XD (U/ ml) XD/XO Total XD/XO Inside AlP (U/ml) AcP (U/ml)

Intact 1.11 - - - 114 16.2

Total 1.55 1.10 0.71 2.5 118 15.1

Distribution of enzymes in MFGM with and without plasmin

deactivation

With plasmin deactivation Without plasmin deactivation

XO (U/g) 2.4 ± 0.4 3.7 ± 0.6

XD (U/g) 1.7 ± 0.5 -

XD/XO Total 0.7 ± 0.08 -

XD/XO inside 4.25 ± 0.5 -AlP (U/g) 135 ± 10 142 ± 15

AcP (U/g) 39 ± 7 42 ± 9

Distribution of protein in milk fractions

Total protein Casein Protein in WMP Protein in MFGM

g/l % of total g/l % of total g/Kg % of total g/Kg % of total

29.2 100 22.7 78 44.3 0.094 40.3 0.095

Lipid composition (as % of dry matter) in whey membrane particles (WMP)

and milk fat globule membranes (MFGM)

WMP MFGM

Average SD Average SD

Total lipid 25.6 2.7 29.7 3.1

Lipid P 0.530 0.09 0.621 0.11

Phos.lipids 13.25 2.25 15.53 2.75

Question Number 3

Does XO-derived oxidative stress play a role in sub-clinical mastitis; i.e., under conditions that do not elicit an apparent

classical inflammatory symptoms

+ -

The model: Each cow tested had at least one uninfected quarter (NBF) and one of the other quarters infected with one of the following bacteria:

BacteriaNumber

NBF33

Streptococci23

CNS11

E. Coli3

S. aureus9

Cork 2005

Uric acid and nitrate in sub-clinically infected glands

BacteriaUric Acid (micro-molar)

Nitrate(micro-molar)

NBF 35 ± 13a 19 ± 9a

Strep. DG 72 ± 14b 38 ± 12b

CNS 38 ± 14a 17 ± 11a

E. coli 85 ±15b 42 ± 12b

S. aureus 39 ± 19a 20 ± 11a

BOLFA 2006

Clotting time and curd firmness

BacteriaClotting time (sec)

Curd firmness(V)

NBF 650±63 6.58±0.2Strep. 2490±340 1.02±0.3CNS 1255±468 3.80±0.8E. coli2590±370 0.92±0.3

S. aureus 1078±193 3.28±0.7

Cork 2005

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Strep. dysgalactiae S. aureus

Curd firmness (A30 Optigraph)

log

Som

atic

Cel

l Cou

nt

Question Number 4

Does XO-derived oxidative stress play a role in clinical mastitis; i.e., under

conditions that elicit an apparent classical inflammatory symptoms

The model: Each cow tested was infused in one quarter once with

Casein hydrolyzate, lipopolysaccharide,or saline, and samples from each gland

were sampled for two days post-treatment

BOLFA 2006

Effect of infusion of CNH and LPS into the mammary gland on the

immune cell population

Treatment SCC (×1000) PMN (%) CD4+ (% CD8+ (%) CD14+ (%)

Control 116±20a 29±3.3a 3.1±0.9a 5.7±1.6a 5.5±1.8a

CNH 3146±324b 57±7b 3.3±1.1a 10.5±2.0b 12.6±2.2b

LPS 4960±793c 90±9.1c 1.8±2.2b 4.4±4.0a 6.6±4.4a

Caseinolysis (proteose peptone formation) in CNH and LPS treated

glands

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0 +24 +48

Time relative to treatment

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LPS

CNH

CONTROL

Uric acid in CNH / LPS treated glands

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Time relative to treatment

Uri

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id u

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LPS

CNH

CONTROL

Nitrate in CNH and LPS treated glands

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Time relative to treatment

Nit

rate

un

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LPS

CNH

CONTROL

Major conclusions

• Our data suggest that XO is post-transcriptional regulated through allocation of substrate (xanthine) availability.

• Together with lactic peroxidase they involve in the oxidative (mostly nitrosative) stress in certain type of sub-clinical mastitis.

• This system is the main driving force of oxidative/nitosative stress in E.Coli/LPS driven mastitis.

The Jekyll and Hyde sides of uric acid

• Uric acid is a major anti-oxidant in blood plasma and milk

• However, uric acid is also a danger signal that alerts the immune system to dying cells (Nature 425: 516, 2003).

• In hyperuricemia, crystals of uric acid can precipitate in joins, where they cause gout and/or in other tissues causing inflammation.

• Does XO-depended gouty inflammation involve in the pathogenesis induced by E. coli/LPS in the mammary gland ?

Thank you: I hope that this lecture

will contribute to our ability to

raise healthier cows and produce

better dairy products

BOLFA 2006