Transcript Uric Acid - PublicationsList.org

Physiological
roles of milk
enzymes: an
evolving picture
Nissim Silanikove
Department of Food Science
Agricultural Research Organization,
The Volcani Center, Israel
Milk phases
Role Of Milk
In Regulation Of Milk
Secretion:
Negative Feed Back Mechanism
Induced By Milk Components
Occasional changes in gland
emptying
Daily changes in breast
volume
Daily changes in breast milk
synthesis
Plasminogen/Plasmin System
Milk plasminogen and plasmin
concentrations (throughout lactation)
Milk plasmin x Milk yield x bST
(throughout lactation)
Stress and the plasmin system
The effect of dexamethasone on the PPS system in cows
Control
Treatment
PA (units/ml)
11.8±0.1
*17.3±0.2
Plasmin (units/ml)
5.42±0.9
*8.75±0.8
Plasminogen (units/ml)
27.3±1.4
24.8±2.1
Plasminogen/Plasmin
5.03±0.8
*2.83±0.9
Values are mean ± SE; *P < 0.05 by t-test
Silanikove et al, Life Sci., 2000
The Negative Feedback Mechanism
Flow-Chart: The ARO View.
Blue arrows denote flow of signal along the feedback loop, red arrows denote positive
effect and black arrows denote suppressive effect
Milk yield (half) of sheep or goat infected
with CNS specie in one gland and the
contra-lateral being free.
Sheep – open bars
2.0
Milk yield (kg/day)
Goats – hatched bars
1.5
1.0
0.5
0.0
Uninfected
Leitner et al., JDS, 2004
Infected
Plasmin activity: sheep or goat with one
gland infected with CNS specie and the
contra-lateral being free
Sheep + 73.7%, P < 0.0007
Goat + 195%, P < 0.0003
60
50
40
Sheep
Goat
30
20
10
0
Uninfected
Leitner et al, JDS, 2004
Infected
Proteose-peptone concentration:
sheep or goat with one gland infected with
CNS specie and the contra-lateral being free
Sheep + 247%, P < 0.0001
Goat +151%, P < 0.0001
2.5
2
1.5
Sheep
Goat
1
0.5
0
Uninfected
Leitner et al, JDS, 2004
Infected
Conclusions
• The basal level of PL activity is higher in
sheep than in goats, which explains the higher
basal level of proteose-peptone
• PL activity in infected glands is higher in
sheep than in goats, which explains the higher
increase in proteose-peptone
• The higher increase in proteose-peptone
concentration in sheep than in goats explains
the more acute reduction in milk yield in sheep
Role Of The Plasmin
System In Induction Of
Active Involution
What happens in case of
surplus?
Involution
Definition
Involution
The model:
Each goat or cow were Injected
with casein hydrolyzate in the experimental
gland (+) whereas the control gland (-) was
treated with intact casein
+
-
CNH in Goats:Multiple treatments
Silanikove et al, Life Sci., 2002
Histology of section in the alveolus
control
CNH treated glands.
Most of the cells stained in the CNH treated gland
are leukocytes
Silanikove et al, unpublished data
Milk Enzymes As Components Of
The Innate Immune System:
Formation Of Free Radicals And
Bacterocidic Bacteristatic Environment
During Active Involution
The many faces of XOR
Nitrate
Nitrite
Or 2H2O2
SOD
Reaction of Lactoperoxidase with
Hydrogen peroxide and Nitrite
1. LPO compound I + H2O2
2. LPO compound I + NO2
-
3. LPO compound I + NO2
-
LPO compound 1
LPO compound II + ●NO2
LPO + ●NO2
Silanikove et al, FRBM, 2005
Silanikove et al, FRBM, 2005
Silanikove et al, FRBM, 2005
Scenario of NO cycling and
metabolism in mammary secretion
Glutathione cycle in milk
NO
Nitrite
LPO
**
*22
Supply to the young
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 )
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
XOR
is
mostly
associated with milk
serum
as
XO,
though its activity is
highest
on
membranes
and
where
it
is
distributed between
a. Physiologically inactive
XD in the inner side of
MFGM and vesicles, and
b. Physiologically active in
the form of XO, on the
outside surface of the
vesicles.
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.
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
Disappearance of hypoxanthine
and appearance of uric acid in
fresh milk
Scenario of NO cycling and
metabolism in mammary secretion
Question Number 3
Does Catalase really plays a critical role in
protecting the gland from oxidative stress
by converting active nitrite to less active
nitrate?
Xanthine dose-dependently enhance the
conversion of nitrite to nitrate.
45
40
No xanthine
Nitrite concentration, M
35
10 M xanthine
30
25
50 M xanthine
20
15
10
100 M xanthine
5
150 M xanthine
0
-5
0
10
20
30
40
50
60
Time, min
Under the experimental conditions, approximately 40 mM of
xanthine are converted to urate via XO within 4 h
Silanikove et al, unpublished data
50
Xa-0
Xa-10
Xa-50
Xa-100
Xa-150
M
40
30
20
10
0
Lg10 Nitrite Conc., M
0
1.8
20
40
60
80
Time, Min
1.6
1.4
1.2
1.0
rate constant min -1 x 1000
0
10
20
30
40
Time, Min
20
15
10
5
0
0
50
100
150
Xanthin concentration, M
200
Catalase inhibitor, amino triazole, prevent the
conversion of nitrite to nitrate.
Conversion of nitrite to nitrate, %
90
80
70
60
50
40
30
20
0
5
10
15
20
Concentration of catalase inhibitor, mM
reaction conditions Nitrite-50 mM, Xanthine-200 mM, incubation time - 30 min.
Silanikove et al, unpublished data
600
1/v
400
-40
200
-20
20
3-Amino triazole, mM
-200
40
Relative changes in lipid oxidation in milk
Relative changes in lipid oxidation, %
160
140
120
100
80
60
40
20
0
A
B
C
D
Treatments
milk stored for 6 hours in the dark at 40C (A), Effects of catalase inhibitor (B), nitrite
(10 mM) (C) and nitrite + catalase (D) inhibitor
Silanikove et al, unpublished data
400
nmole g-1
300
200
100
0
3.5
0
1
2
3
4
5
Time, Days
mEqv g-1
3.0
2.5
2.0
1.5
0
1
nmole g-1
1500
2
3
4
5
Time, Days
1000
500
0
0
1
2
3
Time, Days
4
5
Conclusions Regarding the Control of
Oxidative Stability in Milk
• XO and catalase works interactively as an antioxidant
system.
•
Formation of nitric 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 availability. The supply of the latter at required
physiological rate may be provided from dissolved
oxygen via XO by using electrons gained during the
conversion of xanthine to urate.
Question Number 4
Does XO-LPO 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
8
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 5
Does XO-LPO 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
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 ?
Concluding Remarks
• Milk enzymes have an important biological role and
are involved in control of milk secretion,
developmental stage (involution), gland innate
immune system and preventing oxidative damage to
its essential nutrients.
• For that purpose milk congregate many enzymes
which constantly consume metabolites, produce freeradicals and modify its composition if needed.
• Milk enzyme along with other components (e.g.,
cytokines, enzyme inhibitors) form complex metabolic
pathways.
Thank you: I hope that this lecture
will contribute to our ability to
raise healthier cows and produce
better dairy products
BOLFA 2006