Does Immune Activation Alter Growth Potential and Nutrient Digestibility? - Dr. Jeffrey Escobar, Novus, from the 2014 Allen D. Leman Swine Conference, September 15-16, 2014, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2014-leman-swine-conference-material

1. Alterations in amino acid
digestion and metabolism
during disease
Jeffery Escobar, Ph.D.
Executive Manager
Nutritional Physiology Research

2. Agenda
• Immune activation and metabolism alteration
• Plasma amino acids
– Changes during LPS challenge and their balance
• Amino acid digestibility
– Apparent, basal ENL, standardized
– Salmonella challenge
– Fate of undigested CP/AA
• Conclusions

3. LPS induce inflammatory response
• Inflammatory cytokines
– TNF-α, IL-1b, IL-6
• Induce fever
• Reduce feed intake
• Stimulate
– Skeletal muscle proteolysis
– Liver protein synthesis
• Produce acute phase proteins
Kawai and Akira, 2010

4. Protein synthesis and accretion in
immune challenged pigs
* P < 0.05
Orellana et al., 2002; Escobar et al., 2004

5. Immune activation and metabolism

6. Excess release of AA and nitrogen from skeletal muscle
protein over incorporation into mixed acute-phase proteins
mg/kg BW
AA Nitrogen
Phe 0 0
Trp 2 <1
Val 10 5
Ser 11 1
Cys 12 1
Tyr 16 1
Pro 25 3
Met 26 2
Thr 28 4
Gly 39 7
Ile 41 4
Asp+Asn 61 12
Ala 66 10
His 68 11
Leu 72 8
Arg 83 28
Lys 104 20
Glu+Gln 140 15
– Increase in plasma urea nitrogen
Reeds et al., 1994
• Compare AA composition of skeletal muscle
protein and a mixture of acute-phase
proteins
• Assuming a typical increase in the synthesis
of a mixture of acute-phase protein of 850
mg/kg BW
• The amount of skeletal muscle protein
needed to be mobilized = 1,980 mg
• A difference of 1,150 mg/kg BW
• For a 100 kg pig:
– About 200 g of muscle protein mobilized
– About 13 g of excess nitrogen excreted

7. LPS, cytokines, and plasma urea N
Plasma IL-6, pg/mL
Plasma urea nitrogen, mg/dL
Hour after-injection Hour after-injection
Webel et al., 1997
12-h feed-deprived pigs

8. Immune activation in fasted vs fed state
• Most acute immune activation studies with LPS have
been conducted in the fasted state
– Normal food deprivation period: 8 - 24 h
• Although sick, pigs normally continue to eat, digest,
and absorb nutrients
• Does acute immune activation in the fasted and fed
state result in similar changes
– Digestion, absorption, metabolism, excretion
Arrival
24 h
LPS
Feed
-10 0
Surgery
Fasting Blood sampling
Recovery
-2

9. Blood urea nitrogen in fed pigs
Price, 2011

10. Changes in plasma Phe with LPS
Price, 2011

11. Changes in plasma Phe and urea
nitrogen with LPS in fed pigs
Price, 2011

12. Changes in plasma amino acids with LPS
Price, 2011

13. Area under
the curve
Price, 2011

14. Area under the curve
Price, 2011

15. Estimating plasma amino acid balance
• Calculate the area under the curve for each AA
– Saline and LPS treated pigs
• Use correction factor of 0.075 L plasma/kg BW
to convert AUC to μmolekg BW-1h-1 as a unit
of AA metabolism (Talbot and Swenson, 1970)
• Apply the following equation:
AA balance = (AUCLPS- AUCSAL) 0.075 L 1
kg BW h × × 2
Price, 2011

16. Negative AA balance: SAL > LPS
Price, 2011

17. Positive AA balance: LPS > SAL
Price, 2011

18. Summary of AA metabolism
• Acute immune activation via LPS
– Induce similar changes in plasma urea nitrogen
during the fasted and postprandial state.
– Reduces the digestion and/or absorption of
dietary nutrients or increases AA catabolism
– Alters plasma balance of amino acids
• Lysine was the most negative
• Alanine was the most positive
Is lysine the most limiting AA during immune activation?

19. Phe to Tyr conversion
Harper’s Biochemistry; Kilani et al., 1995; Hsu et al., 2006

20. Changes in plasma Tyr with LPS
Price, 2011

21. Changes in plasma Phe to Tyr
Price, 2011

22. Amino acids:
nutritional considerations
• Dietary amino acids:
– Essential, conditional, nonessential
• “One which cannot be synthesized by the animal
organism out of materials ordinarily available to
the cells at a speed commensurate with the
demands for normal growth” W.C. Rose, 1946
• Essentiality of amino acid for protein synthesis
– Which one is the most limiting AA?, second?
Rethink AA limitation order for metabolic purposes

23. Digestibility vs. bioavailability
van de Waterbeemd and Gifford, 2003

24. Factors affecting AA digestibility
Endogenous
Factors
Species
Breed/strain
Gender
Physiological
state
Exogenous
Factors
Dietary AA
balance
Anti-nutritional
factors
Intake
Pathogens,
disease?

25. Salmonella challenge in pigs
• Salmonella Typhimurium DT104
– Nalidixic acid: 25 mg/mL
– Novobiocin: 20 mg/mL
• Intra-nasal inoculation 9.8×109 cfu
• Euthanized pigs (n=12) 24 and 72 h after oral
inoculation to collect ileal digesta samples
Lee, 2012

26. Apparent ileal AA digestibility, %
24 h after Salmonella inoculation
Lee, 2012

27. Basal endogenous AA losses
24 h after Salmonella inoculation
Lee, 2012

28. Standardized ileal AA digestibility, %
24 h after Salmonella inoculation
Lee, 2012

29. Salmonella challenge in ileal
cannulated pigs
• 8 growing pigs
• Ileal cannulation and recovery
• Intra-nasal inoculation 1.3×109 cfu
Salmonella Typhimurium DT104 (NalRNovR)
• Collect ileal digesta every 8 h from
0 to 72 h after inoculation
Lee, 2012

30. Dynamic changes in AA digestibility in
Salmonella challenged pigs
Lee, 2012

31. Dynamic changes in AA digestibility in
Salmonella challenged pigs
Lee, 2012

32. Summary on digestibility and disease
• Enteric disease results in:
– Lower apparent AA digestibility
– Higher endogenous losses of AA
• Higher gut demand for nutrients and energy
– Production of mucins, digestibe enzymes,
enterocytes, etc.
• Provide highly digestible dietary proteins
• Can poor digestion leads to enteric disease?

33. Anti-nutritional effect of
indigestible protein in the hindgut

34. Hindgut AA fermentation
Meijers and Evenepoel 2011; Wikipedia

35. Protein fermentation metabolites
limiting pig growth
Yokoyama et al., 1982

36. Dietary protease improves
intestinal environment
Dietary protease reduces protein fermentation and
prevents increase in hindgut pH.
Wang et al., 2011

37. Dietary protease improves
intestinal environment
Protease limits growth of undesirable bacteria and
enhances growth of beneficial bacterial species.
Wang et al., 2011

38. General conclusions
• Immune activation alters digestion, absorption,
and metabolism of amino acids
• Fermentation of undigested proteins produce
undesirable bacterial metabolites and alter gut
environment and microbiota
• Feed pigs highly digestible ingredients or use
available technologies to maximize AA digestibility
and hence minimize protein fermentation

39. Acknowledgements
• Students and technicians
– Dr. Kathryn Price
– Dr. Hanbae Lee
– Dr. Matthew Utt
– Elizabeth Ramirez
– Heather Totty
– Greg van Eyk
– Courtney Klotz
– Heather Reeves
– Pat Williams
• Collaborators
– Dr. Rodney Johnson
– Dr. David Baker
– Dr. William van Alstine
– Dr. Teresa Davis
– Dr. Monica Ponder
– Dr. Mark Hanigan
– Dr. Allen Harper
– Dr. Kevin Pelzer
• Virginia Tech Pratt Fellowship

40. Благодаря ви
Obrigado
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Tak! Danke Schoen!
Thank You!
Merci
Gracias
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谢谢
Mulţumesc
Asante sana
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