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

Nitrite NADH NitrateNAD Or 2H2O2 SOD

N=O Reaction of Lactoperoxidase with Hydrogen peroxide and Nitrite Nitric Oxide is a Free Radical 1. LPO + H2O2 LPO compound 1 2. LPO compound I + NO2- LPO compound II + ●NO2 3. LPO compound II + NO2- LPO + ●NO2 N=O

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

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 )

Xanthine + hypoxanthine and uric acid concentration in oxytocin-induced and 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 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: Number Bacteria 33 NBF 23 Streptococci 11 CNS 3 E. Coli 9 S. aureus + - Cork 2005

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

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

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

Uric acid in CNH / LPS treated glands

Nitrate in CNH and LPS treated glands

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