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The impact of dietary methyl donors on whole-body methionine partitioning in the neonatal piglet Jason L Robinson1, Laura E McBreairty1, Scott V. Harding2, Janet A Brunton1, Robert F Bertolo1 1Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada of Nutrition Sciences, School of Medicine, Kings College London, London, UK Background Results • Methionine is an essential amino acid that is required for protein synthesis and it also provides the body with methyl groups (Figure 1) • Remethylation occurs by the dietary methyl donors folate and choline (via betaine) • The dietary intake of methyl donors and methionine are variable during early life; and potentially impact the methionine requirement for protein synthesis Study Protocol A CO2 O H 2N C Tracer oxidized when an amino acid ie. methionine is limiting protein synthesis OH CH2 1.5 4 1.0 2 0.5 0 4 Tracer lost to protein if its not limited by other amino acids (to maximum) 5 6 A 0.0 400 200 0 H 2N O CH C • Full complement of amino acids • Soybean oil as lipid source • Added betaine and choline • Full vitamin complement • Methionine level restricted to 80% of the requirement to limit available methyl groups (D5) CH C OH CH2 SAHH OH CH2 CH2 Adenosine CH2 Adenosine SH H2O + – M M M et et hy l hy l hy l et et M µmol Met/(kg•hr) µmol Phe/kg.hr hy l et hy l – hy l + + – et hy l M et M * 15 10 5 0 p = 0.01 * p = 0.01 Transsulfuration Cystathionine CGL Cysteine 100 S Homocysteine CBS 200 0 S-adenosylhomocysteine (SAH) Transmethylation 20 * 300 M Methyl Deficient (MD-) Diet Methyl Replete (MS+) Diet 400 + O C Breakdown hy l Choline 0.0 p = 0.02 C DNA Betaine 0.5 0 et Creatine 1.0 M Phosphatidylcholine H 2N Table 1: Piglets were randomized to a methyl deficient (MD-) or methyl replete (MS+) diet • Full complement of amino acids • Soybean oil as lipid source • No choline or betaine • No folate • Methionine level restricted to 80% of the requirement to limit available methyl groups (D5) Transmethylation 1.5 et Remethylation 5-MTHF 200 M BHMT MS 5,10-MTHF hy l – Day 7 • Animals received primed constant infusion of [13C, 2H-methyl] Methionine (8 hrs) • Blood and breath samples taken to measure methionine cycle activities Figure 2: Time-course values of plasma (MPE) and breath (APE) enrichment after (A) a 6-hour phenylalanine infusion and (B) an 8-hour methionine infusion 2.0 * 400 – S CH3 OH Time (hours) 600 hy l CH2 0.1 µmol Met/(kg•hr) C CH2 8 Transsulfuration + CH Transmethylation 7 hy l H 2N Transmethylation 6 * p = 0.03 B Synthesis (non-oxidative losses) M THF 0.2 2 p = 0.01 B et Day 6 • Animals received primed constant infusion of [13C] Phenylalanine (6 hrs) • Blood and breath samples taken to measure Phenylalanine flux • Protein synthesis and breakdown calculated stochastically 4 [13C] Met [2H] Met [13C] Hcy APE M OH 0.3 – C CH2 CH3 0.4 6 0 S-adenosylmethionine (SAM) DMG O 8 µmol Phe/kg. hr CH Days 1-5 H 2N S+ CH3 8 hours O CH2 Adenosine Methionine Plasma Enrichment MPE MAT CH3 13CO 2 5 0 hy l 6 hours 13CO 2 OH CH2 0.5 et 13CO 13CO 13CO 13CO 2 2 2 2 CH C 10 Breath Enrichment APE H 2N PPi + Pi ATP S 13CO 13 CO2 13CO2 13CO2 13CO2 2 B O OH CH2 CH2 13CO 2 * 10 Time (hrs) O CH C H 2N Remethylation 15 * • Animals were divided into two groups based on diet (Table 1) • All animals received two enteral isotope infusions; breath and blood samples were taken for MS analysis A Phenylalanine Flux 600 APE MPE et Protein Synthesis 2.0 6 + • Diet was chronically infused using TPN pumps into the gastric catheter for the duration of the experiment CH 8 Breath Enrichment APE • 3-8 day old Yucatan miniature pigs were anesthetised and surgically implanted with gastric and venous catheters µmol Met/(kg•hr) • As a result the methionine requirement is changed in the presence or absence of dietary cysteine µmol Phe/(kg•hr) • Demethylated methionine (homocysteine) can either form cysteine by “Transsulfuration” or be reformed by “Remethylation” Plasma [13C] Phe Enrichment MPE • Phenylalanine and methionine infusions reached plateau in blood and breath in 4-, and 6-hrs respectively (Figure 2) • Stochastic measures of the phenylalanine infusion resulted in a number of differences based on dietary methyl donors (Figure 3) • Phenylalanine flux was greater with methyl donors (MD- 425.7 ± 46.5 µmol Phe/(kg•hr) vs. MS+ 488.8 ± 32.6 Phe/(kg•hr); p=0.01) • Protein Synthesis was significantly higher with methyl donors (MD- 424.6 ± 46.7 µmol Phe/(kg•hr) vs. MS+ 480.7 ± 36.6 Phe/ (kg•hr); p=0.02) • Protein Breakdown was lower during methyl deficiency (MD- 221.7 ± 44.4 µmol Phe/(kg•hr) vs. MS+ 282.8 ± 34.81 Phe/ (kg•hr); p=0.01) • The flux of methionine through remethylation, transsulfuration and transmethylation are depicted in Figure 4: • Remethylation was higher in the MS+ piglets (MD- 5.1 ± 5.0 µmol Met/(kg•hr) vs. MS+ 12.8 ± 6.0µmol Met/(kg•hr); p=0.03) • Transsulfuration was unchanged between the two groups and was highly variable during methyl deficiency • Overall Transmethylation was enhanced in the presence of methyl donors (MD- 5.4 ± 5.0 µmol Met/(kg•hr) vs. MS+ 14.6 ± 5.9 µmol Met/(kg•hr); p=0.01 • Methyl groups are used in “Transmethylation” to synthesize critical metabolites and regulate gene expression through DNA methylation M 2Division Expired CO2 Figure 1: A schematic of the Methionine cycle. Note that isotope positions are highlighted for the methionine infusion. Objectives • We aimed to quantify the potential for dietary methyl donors to affect the methionine requirement for protein and transmethylation • To do this we restricted dietary methionine in neonatal piglets and compared in vivo methionine metabolism in individuals with- and without dietary methyl donors • It was hypothesized that methyl donors would affect protein synthesis, remethylation and transmethylation Figure 3: After a 6-hour phenylalanine infusion breath and blood samples were used to calculate phenylalanine flux (A), protein synthesis (B) and protein breakdown (C). Figure 4: Rates of (A) Remethylation, (B) Transsulfuration and (C) Transmethylation with and without dietary methyl donors in methionine restricted piglets Discussion • Dietary methyl donors had a significant impact on whole-body protein dynamics in neonatal piglets • Whole-body protein synthesis and breakdown were enhanced by dietary methyl donors by a similar amount, although protein synthesis was quantitatively greater overall • The enteral flux of dietary phenylalanine was impacted significantly in the presence of methyl donors • Through an increase in remethylation, methyl donors “spared” methionine for transmethylation and protein metabolism • In conclusion dietary methyl donors should be considered when determining the methionine requirement in the neonate