Piglets Are Vulnerable to Intestinal Inflammation and Infection Because of Expression of Low‐Affinity Jejunal Alkaline Phosphatase Activities

Intestinal alkaline phosphatase (IAP) plays a pivotal role in protecting gut health through catalyzing the hydrolytic dephosphorylation of the lipid A moiety of endotoxin lipopolysaccharides (LPS) and other emblematic members of pathogen‐associated‐molecular patterns (PAMPs) such as ATP, ultimately detoxifying endotoxins and preventing gut dysbiosis and disorders. Characterization of IAP hydrolytic activities, including enzyme affinity ( K m ) and the maximum enzyme activity ( V max ), has been reported in pigs and other mammalian species by using the chromogenic synthetic substrate P ‐nitrophenyl phosphate (pNPP) rather than using relevant physiological substrates such as ATP and LPS under the physiological pH conditions. This study was conducted to determine kinetics of young porcine jejunal alkaline phosphatase in hydrolyzing the dephosphorylation of ATP, the lipid A moiety of LPS and pNPP at pH 7.4 and 37 °C. Jejunal samples were collected from four 10‐day old Yorkshire pigs and were pulverized to be homogenous under liquid N 2 . The LPS kinetic experiments were conducted with 11 gradient concentrations of LPS, isolated from E. coli 0111:B4, ranging 0 – 5 mg/mL in incubation media. The ATP kinetic experiments were conducted with 11 gradient concentrations of ATP, ranging 0 – 5 mM in incubation media. Free phosphorus (Pi) release was measured by using the PiColorLock detection reagent (Innova Biosciences). Contributions of the non‐specific background Pi associated with ATP and LPS in the time course and the kinetic experiments were corrected. The pNPP kinetic experiments were conducted with 16 gradient concentrations of pNPP, ranging 0 – 6 mM in incubation media. All enzymatic incubations were conducted with 19 – 27 μg of the homogenized porcine jejunal protein. The kinetics (parameter estimates ± SE, P < 0.05) of young porcine jejunal alkaline phosphatase activities were obtained, including V max values of 2.5 ± 0.4 ug/mg protein·min (n = 11), 1.1 ± 0.2 ug/mg protein·min (n = 11), and 0.12 ± 0. 01 umol/mg protein·min (n = 16); and K m values of 1.255 ± 0.498 mM (n = 11), 2.388 ± 0.794 mg/mL (n = 11), and 0.145 ± 0.036 mM (n = 16), for the hydrolysis of ATP, LPS and pNPP, respectively. Our results reveal the young porcine IAP K m value 41 times higher in comparison with reported K m value of human IAP in dephosphorylating ATP, 3 times higher when compared with reported human IAP dephosphorylating LPS, and 5 times higher when compared with reported human IAP hydrolyzing pNPP. Results of this study show that young porcine jejunal IAP activity affinity is several fold lower in detoxifying endotoxin LPS the other emblematic PAMPs member ATP in comparison with humans, suggesting that young pigs are susceptible to gut inflammation and infection. Support or Funding Information NSERC Discovery Program of Canada and Metagen Enzyme Corporation