Physiological Physiological roles of milk roles of milk enzymes: an enzymes: an

evolving pictureevolving picture

Nissim SilanikoveNissim SilanikoveDepartment of Food Science

Agricultural Research Organization,The Volcani Center, Israel

Milk phasesMilk phases

Role OfRole Of Milk Milk

In Regulation Of Milk In Regulation Of Milk

Secretion:Secretion:

Negative Feed Back Mechanism Negative Feed Back Mechanism Induced By Milk ComponentsInduced By Milk Components

Occasional changes in gland Occasional changes in gland emptyingemptying

Daily changes in breast Daily changes in breast volumevolume

Daily changes in breast milk Daily changes in breast milk synthesissynthesis

Plasminogen/Plasmin SystemPlasminogen/Plasmin System

Milk plasminogen and plasmin Milk plasminogen and plasmin concentrations concentrations (throughout lactation)(throughout lactation)

Milk plasmin x Milk yield x bSTMilk plasmin x Milk yield x bST (throughout lactation)(throughout lactation)

Stress and the plasmin systemStress and the plasmin system

Silanikove et al, Life Sci., 2000

ControlTreatment

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±1.424.8±2.1

Plasminogen/Plasmin5.03±0.8*2.83±0.9

The effect of dexamethasone on the PPS system in cowsThe effect of dexamethasone on the PPS system in cows

Values are mean ± SE; *P < 0.05 by t-test

The Negative Feedback Mechanism The Negative Feedback Mechanism Flow-Chart: The ARO View.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 Milk yield (half) of sheep or goat infected with CNS specie in one gland and the with CNS specie in one gland and the

contra-lateral being free.contra-lateral being free.

0.0

0.5

1.0

1.5

2.0

Uninfected Infected

Milk y

ield

(kg

/da

y)

Leitner et al., JDS, 2004

Sheep – open bars

Goats – hatched bars

0

10

20

30

40

50

60

Uninfected Infected

Sheep

Goat

PL

activ

ity,

uni ts

/mL

Plasmin activity:Plasmin activity: sheep or goat with one sheep or goat with one gland infected with CNS specie and thegland infected with CNS specie and the

contra-lateral being freecontra-lateral being free

Sheep + 73.7%, P < 0.0007Goat + 195%, P < 0.0003

Leitner et al, JDS, 2004

0

0.5

1

1.5

2

2.5

Uninfected Infected

Sheep

Goat

P-p

, g /

L

Proteose-peptone concentration:Proteose-peptone concentration: sheep or goat with one gland infected with sheep or goat with one gland infected with

CNS specie and the contra-lateral being freeCNS specie and the contra-lateral being free

Sheep + 247%, P < 0.0001Goat +151%, P < 0.0001

Leitner et al, JDS, 2004

ConclusionsConclusions

• The basal level of PL activity is higher in The basal level of PL activity is higher in sheep than in goats, which explains the higher sheep than in goats, which explains the higher basal level of proteose-peptonebasal level of proteose-peptone

• PL activity in infected glands is higher in PL activity in infected glands is higher in sheep than in goats, which explains the higher sheep than in goats, which explains the higher increase in proteose-peptoneincrease in proteose-peptone

• The higher increase in proteose-peptone The higher increase in proteose-peptone concentration in sheep than in goats explains concentration in sheep than in goats explains the more acute reduction in milk yield in sheepthe more acute reduction in milk yield in sheep

Role Of The Plasmin Role Of The Plasmin

System In Induction Of System In Induction Of

Active InvolutionActive Involution

What happens in case of surplus?What happens in case of surplus?

InvolutionInvolutionDefinitionDefinition

InvolutionInvolution

The model:The model:

Each goat or cow were InjectedEach goat or cow were Injectedwith casein hydrolyzate in the experimentalwith casein hydrolyzate in the experimentalgland (+) whereas the control gland (-) was gland (+) whereas the control gland (-) was treated with intact caseintreated with intact casein

+ -

CNH in Goats:Multiple treatmentsCNH in Goats:Multiple treatments

Silanikove et al, Life Sci., 2002

CNH treated glands. CNH treated glands.

Silanikove et al, unpublished data

Histology of section in the alveolus Histology of section in the alveolus

Most of the cells stained in the CNH treated gland Most of the cells stained in the CNH treated gland are leukocytesare leukocytes

control control

Milk Enzymes As Components Of Milk Enzymes As Components Of

The Innate Immune System:The Innate Immune System:

Formation Of Free Radicals And Formation Of Free Radicals And Bacterocidic Bacteristatic Environment Bacterocidic Bacteristatic Environment

During Active InvolutionDuring Active Involution

Or 2H2O2

Nitrate Nitrite

SOD

The many faces of XOR The many faces of XOR

Reaction of Lactoperoxidase with Reaction of Lactoperoxidase with Hydrogen peroxide and NitriteHydrogen peroxide and Nitrite

1 .LPO compound I + H2O2 LPO compound 1

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

3 .LPO compound I + NO2- LPO + ●NO2

Silanikove et al, FRBM, 2005

                                                   

Silanikove et al, FRBM, 2005

Silanikove et al, FRBM, 2005

Scenario of NO cycling and Scenario of NO cycling and metabolism in mammary secretionmetabolism in mammary secretion

LPO

Supply to the young

**

NO

Nitrite

Glutathione cycle in milkGlutathione cycle in milk

22*

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 )

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

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.

XORXOR is mostly is mostly associated with milk associated with milk serum as serum as XOXO, , though its activity is though its activity is highest on highest on membranes and membranes and where it is where it is distributed betweendistributed between

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

0

5

10

15

20

25

30

35

40

45

50

Oxytocin Induced Mature milk

Mic

ro-m

ola

r

Xanthin+HypoxanthinUric Acid

Disappearance of hypoxanthine and appearance of uric acid in

fresh milk

Scenario of NO cycling and Scenario of NO cycling and metabolism in mammary secretionmetabolism 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 Xanthine dose-dependently enhance the

conversion of nitrite to nitrateconversion of nitrite to nitrate . .

0 10 20 30 40 50 60-5

0

5

10

15

20

25

30

35

40

45

150 M xanthine

100 M xanthine

50 M xanthine

10 M xanthine

No xanthine

Nitri

te c

on

ce

ntr

atio

n, M

Time, min

Silanikove et al, unpublished data

Under the experimental conditions, approximately 40 mM of Under the experimental conditions, approximately 40 mM of xanthine are converted to urate via XO within 4 hxanthine are converted to urate via XO within 4 h

0 20 40 60 800

10

20

30

40

50Xa-0

Xa-10Xa-50Xa-100Xa-150

Time, Min M

0 10 20 30 401.0

1.2

1.4

1.6

1.8

Time, Min

Lg 1

0 N

itri

te C

on

c.,

M

0 50 100 150 2000

5

10

15

20

Xanthin concentration, M

rate

co

nsta

nt

min

-1 x

1000

Catalase inhibitor, amino triazole, prevent the Catalase inhibitor, amino triazole, prevent the

conversion of nitrite to nitrate. conversion of nitrite to nitrate.

0 5 10 15 2020

30

40

50

60

70

80

90C

on

vers

ion

of

nitr

ite to

nitr

ate

, %

Concentration of catalase inhibitor, mM

Silanikove et al, unpublished data

reaction conditions Nitrite-50 mM, Xanthine-200 mM, incubation time - 30 min.reaction conditions Nitrite-50 mM, Xanthine-200 mM, incubation time - 30 min.

-40 -20 20 40

-200

200

400

600

3-Amino triazole, mM

1/v

Relative changes in lipid oxidation in milk Relative changes in lipid oxidation in milk

A B C D0

20

40

60

80

100

120

140

160

Re

lativ

e c

ha

nge

s in

lipi

d o

xid

atio

n, %

Treatments

Silanikove et al, unpublished data

milk stored for 6 hours in the dark at 40C (A), Effects of catalase inhibitor (B), nitrite 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(10 mM) (C) and nitrite + catalase (D) inhibitor

0 1 2 3 4 50

100

200

300

400

Time, Daysnm

ole

g-1

0 1 2 3 4 51.5

2.0

2.5

3.0

3.5

Time, Days

mE

qv g-1

0 1 2 3 4 50

500

1000

1500

Time, Days

nm

ole

g-1

Conclusions Regarding the Control of Conclusions Regarding the Control of Oxidative Stability in MilkOxidative Stability in Milk

• 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.

• XO and catalase works interactively as an antioxidant system.

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:

BacteriaNumber

NBF33

Streptococci23

CNS11

E. Coli8

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 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 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

350

400

0 +24 +48

Time relative to treatment

Uri

c ac

id u

nol

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 ?

Concluding RemarksConcluding 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 free-radicals 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