Amino&Acids
Non+Essential&– Ala,&Gly,&Ser,&Asp,&Glu,&Pro,&Arg
Essential&– Leu,&Ileu,&Phe,&Thr,&Val
Lipids
Fatty&Acids
Unsaturated:&16:1&/&18:1&/&18:2(n+6)&/&20:4(n+6)&/&20:5(n+3)
Saturated:&14:0&/&16:0&/&18:0
Cholesterol
Sugars,&Starches,&and&Acids
Sucrose&/&Glucose&/&Malate&/&Pyruvate
Non$Essential,(Dispensables)
Alanine
Glycine
Serine
Aspartic,Acid
Glutamic,Acid
Proline*
Arginine*
Hydroxyproline*
Asparagine
Cysteine*
Glutamine*
Selenocysteine*
Tyrosine*
Histidine*
Ornithine*
Taurine*
*essential,in,some,cases
Essential,(Indispensables)
Isoleucine
Leucine
Phenylalanine
Threonine
Valine
Methionine
Lysine
Tryptophan
(1),Acid,Hydrolysis
~5mg,of,tissue,hydrolyzed,in,6N,HCl at,110˚C,for,20,hours
Glutamine,converted,to,glutamic,acid,during, hydrolysis
(2),Esterification,of,Carboxyl,Terminus
Add,2$3ml,of,1:4,acetyl,chloride:isopropanol
Heat,at,110˚C,for,60,minutes
Adding, C,(and,H),,But,We,Know
Where,,How,Much,,and,δ value
CH3COCl
(3),Trifluoroacetylation of,Amine,Group
Add,2$3ml,of,3:1,dichloromethane:trifluoroacetic anhydride,(TFAA)
Heat,at,110˚C,for,15,minutes
Amine
Group
Carboxyl
Group
C4F6O3
Autosampler
Compound
Combustion
Reduction
Pyrolysis
Compound
Separation
Conflo
Magic/Voodoo
Non+Essential&Amino&Acids
Essential&Amino&Acids
(Ala/Gly/Ser/Glu/Asp/Arg/Pro)
(Val/Leu/Ileu/Phe/Thr)
Carbon&from&Diet
(protein/carbohydrates/lipids)
Carbon&from&Dietary&Protein
Thr
Gly
OR
Gut&Microbes
Glycine
Serine
Alanine
Glucose
3-P-Glycerate
Pyruvate
(1) Glucogenic AAs
Acetyl - CoA
Oxaloacetate
Aspartate
TCA
Cycle
(3) Essential AAs:
Leucine
Glycolysis Isoleucine
Phenyalanine
Threonine
Valine
Lipids
(2) Ketogenic AAs
Glutamine
α-Ketoglutarate
Glutamate
Arginine*
Proline*
Godmother,
$12.4
$25.3
~16‰ Range,in,δ15N,in,1,Pig!
~20‰ Range,in,δ13C,in,1,Pig!
C4 Pigs
C3 Pigs
Similar,ranges/patterns,found,in
modern,and,fossil,,terrestrial,and,marine,,
herbivores,and,carnivores,
Bone$Diet,δ15N,Discrimination,Factors
Amphidon floridensis
O’Brien,,Boggs,,and,Fogel 2002
Adult,(C4 sugar),diet,contributed,≥60%,to,non$essential,AAs,in,hawkmoth eggs;,
essential,amino,acids,sourced,from,larval,(plant),diet,,
Howland,et,al.,2003
Non$Essentials,Glutamate,and,Alanine
track,bulk,diet,δ13C
Essentials,Leucine and,Phenyalanine
track,dietary,amino,acid,δ13C
Compared,Individual,δ13CAA to,Mean,δ13CEAA
Focused,on,3,essential,AAs,(Ileu/Leu/Lys),
whose relative,difference,from,mean,
essential,AAs,changed,among
taxonomic,groups.
Essential
Non$Essential
Dietary
Carbohydrate
Lipid
Carbon
Energy (CO2)
+
Lipid Storage
Dietary
Protein
Carbon
Protein
Tissue Synthesis
Remember,&the&size&of&arrows&depends&on&relative&intake…
Carbohydrates
Newsome&et&al.&2010
Glucose
Serine&&&Glycine
Pyruvate
Alanine
Acetyl
CoA
Carbohydrates
Protein
13
δ C:&+27‰
TCA
Cycle
Oxaloacetate
Aspartate
α!Ketoglutarate
Glutamate
Arginine
Proline
Carbohydrates
Glucose
(+27‰)
Serine&&&Glycine
Pyruvate
Acetyl
CoA
TCA
Cycle
Newsome&et&al.&2010
Carbohydrates
Protein
13
δ C:&+27‰
Oxaloacetate
Lipids
δ13C:&+15‰
Aspartate
Alanine
Lipids
(+15‰)
α!Ketoglutarate
Glutamate
(Arginine&&&Proline)
To&what&degree&do&animals&use&carbon&from&non+protein&dietary&sources
(fat&and&carbs)&to&biosynthesize&proteinaceous tissues?
Weanling,mice,fed,ad/lib/for,4,months,(2–3x,initial,weight)
Bulk,Diets
Lipids
Carbs
C4
Protein
C3
40P:5F
30P:15F
20P:25F
Diet&Treatment
5P:40F
Newsome et,al.,(2014)
Mice&use&lipid&carbon&to&build&non+essential&amino&acids,&especially&Glu and&Asp.
40P:5F
30P:15F
20P:25F
Diet&Treatment
5P:40F
Newsome et,al.,(2014)
Only&protein&carbon&is&used&to&synthesize&mouse&muscle&essential&amino&acids
Total,Non$Essential,%
Total,Essential,%
Muscle
61
39
Amino&Acid
Pathway
Casein
Glycine&(Gly)
Serine&(Ser)
Glycolytic
Glycolytic
Alanine&(Ala)
Aspartate&(Asp)
Glutamate&(Glu)
Proline&(Pro)
Dietary&Lipid&Content
+13.2&(0.8)
+17.0&(0.1)
5%&(6)
+15.3&(0.4)A
+19.2&(0.7)A
15%&(7)
+15.5&(0.5)A
+19.6&(0.8)A
25%&(7)
+15.1&(0.3)A
+18.4&(0.7)A
40%&(6)
+12.1&(0.8)B
+14.0&(1.1)B
Glycolytic/Krebs
Krebs
Krebs
Krebs
+22.1&(0.1)
+24.2&(0.1)
+19.8&(0.1)
+21.3&(0.1)
+21.0&(0.3)A
+22.7&(0.5)A
+18.8&(1.0)A
+18.5&(0.4)A
+20.2&(0.7)A,B
+21.0&(0.3)B
+14.1&(0.2)B
+18.1&(0.3)A
+18.9&(0.7)B
+18.6&(0.4)C
+12.2&(0.7)C
+16.2&(0.4)B
+17.3&(1.3)B
+16.5&(0.9)C
+11.6&(1.6)B,C
+13.9&(1.1)B
Leucine&(Leu)
Isoleucine&(Ileu)
Valine&(Val)
Phenylalanine&(Phe)
Essential
Essential
Essential
Essential
+30.6&(0.1)
+27.3&(0.4)
+29.5&(0.2)
+27.4&(0.1)
+30.1&(0.5)A
+23.8&(0.6)A
+23.9&(0.6)A
+28.2&(0.5)A
+29.1&(0.1)A
+24.0&(0.4)A
+24.5&(0.5)A
+27.1&(0.3)A
+29.3&(0.2)A
+23.5&(0.4)A
+24.6&(0.6)A
+28.0&(1.0)A
+28.9&(0.5)A
+22.9&(0.6)A
+24.0&(1.2)A
+26.8&(0.5)A
Bulk&Muscle
–
–
–
–
–
–
+23.1&(0.7)A
1.7&(0.7)
3.8&(0.7)
+22.8&(0.3)A
0.5&(0.7)
4.1&(0.4)
+21.9&(0.6)B
0.2&(1.1)
5.0&(0.6)
+20.4&(0.4)C
+1.1&(1.5)
6.5&(0.4)
D13CMuscle+NLE&(Bulk)&Diet
D 13CMuscle+LE&Diet
For,TCA,synthesized,AAs,(ketogenic), significant,increases,from,5%,to,15%,dietary,lipid,content.
For,Glycolytic,AAs,(glucogenic), significant,increases,from,25%,to,40%,dietary,lipid,content.,
Glycine
Serine
Alanine
Glucose
3-P-Glycerate
Pyruvate
(1) Glucogenic AAs
Acetyl - CoA
Oxaloacetate
Aspartate
TCA
Cycle
(3) Essential AAs:
Leucine
Glycolysis Isoleucine
Phenyalanine
Threonine
Valine
Lipids
(2) Ketogenic AAs
Glutamine
α-Ketoglutarate
Glutamate
Arginine*
Proline*
C3 (+27‰)
C4 (+11‰)
Kelly&and&Martinez&del&Rio&2010
To&what&degree&does&protein&routing&occur&in&an&omnivorous&ectotherm?
Do&mixing&models,&which&assume&that&macromolecules&are&dissembled&into
elements&and&then&reassembled&into&various&compounds&(e.g.,&amino&acids),
underestimate&the&contribution&of&dietary&protein&to&overall&diet?&&
What&proportion&of&the&indispensable&amino&acid&budget&in&tilapia&is&derived
from&gastrointestinal&microbiota?
?
Kelly&and&Martinez&del&Rio&2010
Suggests&a&large&fraction&of&the&carbon&in&indispensable&amino&acids&in&fish
fed&diets&with&<30%&protein&was&derived&from&carbohydrates&and&lipids.
Glycolytic
TCA&Cycle
Essentials
Glucose
Glycine
(C4 Carbohydrates)
Serine
3+P+Glycerate
Alanine
Pyruvate
Acetyl&+ CoA
TCA
Cycle
Glycolysis
(Acetyl&+ CoA)
Oxaloacetate
Aspartate
Arginine* Proline*
TCA
Cycle
α+Ketoglutarate
Glutamate
Applying&discrimination&factors&from&Larsen&et&al.&2009&for&Actinobacterium
and&Rhodococcus spp.:grown&on&a&sole+sucrose&carbon&source.&
The&incorporation&of&dietary&protein&carbon&was&dependent&on&dietary
protein&content&and&on&each&amino&acids biosynthesis&pathway.&
Glycolytic&amino&acids&(Gly/Ser/Ala)&reflected&a&large&contribution&of&dietary&
carbohydrates&and&a&small&contribution&of&dietary&protein.
TCA&cycle&amino&acids&(Asp/Glu)&paralleled&bulk&diet.
Essential&amino&acids&resembled&that&of&carbohydrates&at&low&protein&intakes&
but&were&similar&to&their&corresponding&dietary&amino&acid&as&protein&intake&
increased.&This&pattern&is&consistent&with&assimilation&of&microbially
synthesized&essential&amino&acids&by&tilapias&fed&low&protein&food.
TAKE&HOME:
(1) Protein&routing&occurs&in&some&dispensable&amino&acids&and&the&
scrambled&egg assumption&of&mixing&models&is&incorrect&for&omnivores.&
(2)&CSIA&is&a&novel&method&to&quantify&the&contribution&of&symbiotic
microbes&to&the&indispensable&amino&acid&budgets&of&animals.&