Transcript Uric Acid - PublicationsList.org

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
Nitrate
NAD
Nitrite
NADH
Or 2H2O2
SOD
Reaction of Lactoperoxidase
with Hydrogen peroxide and
Nitrite
Nitric Oxide is a Free Radical
N=O
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, nonenzymatic, 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
50
45
40
Micro-molar
35
30
25
20
15
10
5
0
Oxytocin Induced
Xanthin+Hypoxanthin
Uric Acid
Mature milk
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
% of total
Whoe milk
100
Fat* (MFGM)
33
WMP
21
Phos.lipids
54
Casein
3
Truly soluble
43
Alkaline phosphatase
% of total
100
45
39
84
16
Acid phosphatase
% of total
100
48
34
82
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
Total
1.55
-
1.10
-
0.71
-
2.5
114
16.2
118
15.1
Distribution of enzymes in MFGM
with and without plasmin
deactivation
XO (U/g)
XD (U/g)
With plasmin deactivation
2.4 ± 0.4
1.7 ± 0.5
Without plasmin deactivation
3.7 ± 0.6
-
XD/XO Total
0.7 ± 0.08
-
XD/XO inside
4.25 ± 0.5
-
AlP (U/g)
AcP (U/g)
135 ± 10
39 ± 7
142 ± 15
42 ± 9
Distribution of protein in milk
fractions
Total protein
g/l % of total
29.2 100
Casein
g/l % of total
22.7
78
Protein in WMP Protein in MFGM
g/Kg % of total
g/Kg % of total
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
Average SD
Total lipid 25.6
2.7
Lipid P
0.530 0.09
Phos.lipids 13.25 2.25
MFGM
Average
29.7
0.621
15.53
SD
3.1
0.11
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:
Bacteria
+
-
Number
NBF
33
Streptococci
23
CNS
11
E. Coli
3
S. aureus
9
Cork 2005
Uric acid and nitrate in sub-clinically
infected glands
Bacteria
Uric Acid (micromolar)
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
39 ± 19a
20 ± 11a
S. aureus
BOLFA 2006
Clotting time and curd firmness
Bacteria
NBF
Strep.
CNS
E. coli
S. aureus
Clotting time
(sec)
650±63
2490±340
1255±468
2590±370
1078±193
Curd firmness
(V)
6.58±0.2
1.02±0.3
3.80±0.8
0.92±0.3
3.28±0.7
Cork 2005
log Somatic Cell Count
1000
Strep. dysgalactiae
S. aureus
100
0
1
2
3
4
Curd firmness (A30 Optigraph)
5
6
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
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
5.5±1.8a
Caseinolysis (proteose peptone
formation) in CNH and LPS treated
glands
6,000
LPS
prot-pept ug/ml
5,000
4,000
CNH
CONTROL
3,000
2,000
1,000
0
+24
Tim e relative to treatm ent
+48
Uric acid in CNH / LPS treated glands
400
Uric acid unol
350
300
LPS
CNH
CONTROL
250
200
150
100
50
0
+24
Time relative to treatment
+48
Nitrate in CNH and LPS treated glands
160
140
Nitrate unol
120
LPS
CNH
CONTROL
100
80
60
40
20
0
+24
Tim e relative to treatm ent
+48
Major conclusions
• Our data suggest that XO is posttranscriptional 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