1. Nissim Silanikove, Fira Shapiro, Adi Rauch & Gabriel Leitner
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

2. Nitrite
NADH
NitrateNAD
Or 2H2O2
SOD

3. N=O Reaction of Lactoperoxidase with Hydrogen peroxide and Nitrite
Nitric Oxide is a Free Radical
1. LPO + H2O LPO compound 1
2. LPO compound I + NO LPO compound II + ●NO2
3. LPO compound II + NO LPO + ●NO2
N=O

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

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

6. Xanthine + hypoxanthine and uric acid concentration in oxytocin-induced and mature milk

7. Question Number 2
As mature fresh milk do not contains measurable amount of xanthine, but contains uric acid in the range of 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.

8. 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
Fat* (MFGM)
WMP
Phos.lipids
Casein
Truly soluble

9. 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
Total

10. Distribution of enzymes in MFGM with and without plasmin deactivation
With plasmin deactivation Without plasmin deactivation
XO (U/g) ± ± 0.6
XD (U/g) ±
XD/XO Total ±
XD/XO inside ±
AlP (U/g) ± ± 15
AcP (U/g) ± ± 9

11. 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

12. 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
Lipid P
Phos.lipids

13. 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

14. 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:
Number
Bacteria 33
NBF 23
Streptococci 11
CNS 3
E. Coli 9
S. aureus + -
Cork 2005

15. Uric Acid (micro-molar)
Uric acid and nitrate in sub-clinically
infected glands
Nitrate
(micro-molar)
Uric Acid (micro-molar)
Bacteria
19 ± 9a
35 ± 13a
NBF
38 ± 12b
72 ± 14b
Strep. DG
17 ± 11a
38 ± 14a
CNS
42 ± 12b
85 ±15b
E. coli
20 ± 11a
39 ± 19a
S. aureus
BOLFA 2006

16. Clotting time and curd firmness
(V)
Clotting time (sec)
Bacteria
6.58±0.2
650±63
NBF
1.02±0.3
2490±340
Strep.
3.80±0.8
1255±468
CNS
0.92±0.3
2590±370
E. coli
3.28±0.7
1078±193
S. aureus
Cork 2005

19. 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

20. 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

21. Effect of infusion of CNH and LPS into the mammary gland on the immune cell population
Treatment SCC (×1000) PMN (%) CD4+ (% CD8+ (%) CD14+ (%)
Control ±20a ±3.3a ±0.9a ±1.6a ±1.8a
CNH ±324b ±7b ±1.1a ±2.0b ±2.2b
LPS ±793c ±9.1c ±2.2b ±4.0a ±4.4a

22. Caseinolysis (proteose peptone formation) in CNH and LPS treated glands

23. Uric acid in CNH / LPS treated glands

24. Nitrate in CNH and LPS treated glands

25. 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.

26. 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 ?

27. Thank you: I hope that this lecture will contribute to our ability to
raise healthier cows and produce
better dairy products
BOLFA 2006