Download The impact of dietary methyl donors on whole

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