Distribution of Xanthine Dehydrogenase and Xanthine Oxidase in milk fractions : Evidence for...
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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
0
5
10
15
20
25
30
35
40
45
50
Oxytocin Induced Mature milk
Mic
ro-m
ola
r
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
100
1000
0 1 2 3 4 5 6
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
-
1,000
2,000
3,000
4,000
5,000
6,000
0 +24 +48
Time relative to treatment
pro
t-p
ept
ug
/ml
LPS
CNH
CONTROL
Uric acid in CNH / LPS treated glands
-
50
100
150
200
250
300
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400
0 +24 +48
Time relative to treatment
Uri
c ac
id u
nol
LPS
CNH
CONTROL
Nitrate in CNH and LPS treated glands
-
20
40
60
80
100
120
140
160
0 +24 +48
Time relative to treatment
Nit
rate
un
ol
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