Download Effects of oral supplementation with L

Effects of oral supplementation
with L-citrulline on recovery from
myocardial infarction in mice
By Kevin Grauberger
Under direction of Dr. Mark Stayton
Molecular Biology
Funded by INBRE
Background
• Since 1900, cardiovascular disease has been
the leading cause of death in every year
except 1918.
• CVD kills more people each year than the next
five leading causes of death combined.
What happens during a heart attack?
1. The left coronary
artery (LCA) becomes
blocked
2. The tissue of the left
ventricle is deprived
of blood
Picture by J. Heuser. Wikimedia Commons
What happens in the left ventricle
during a heart attack?
• Changes in mRNA transcription (Harpster et al.)
– Arginase production goes up
– Nitric oxide synthase production goes down
• This means less arginine is converted to nitric oxide
and is instead converted to ornithine
What happens in the left ventricle
during a heart attack?
PIN
eNOS
iNOS
nNOS
NO+Citrulline
AS
ASL
L-arg
ARG1
ARG2
Urea
Aspartate
Argininosuccinate
Fumarate
Ornithine
ODC
AZI
• Up regulated enzymes
• Down regulated enzymes
Putrescine
SPDS
SSAT
AZ
Polyamines
Question
• Nitric oxide is a vasodilator and improves
contractility
• Nitric oxide production goes down during MI
• Would increased levels of nitric oxide make it
easier to survive a heart attack?
Would increased levels of nitric oxide make it
easier to survive a heart attack?
• Questions to answer
1. Can the concentration of arginine be artificially
increased in the blood? My project was to
answer Question 1.
2. Does a higher concentration of arginine lead to
more nitric oxide in heart tissue?
3. Does the heart function better physiologically as a
result of having more arginine?
This data was evaluated and I will
present some of the results
Q1. Can the concentration of arginine be
artificially increased in the blood?
• Problem: getting dietary arginine past the liver
– This problem was observed by Shulman et al.
• Possibility: give dietary citrulline
Q1. Can the concentration of arginine be
artificially increased in the blood?
Blood flow
Nitric oxide
Arginine
Citrulline
Heart
Stomach
Citrulline
Intestine
Liver
Argininosuccinate
Arginine
Experimental
Samples
Number of mice
in each group
Day 28
Heart attack
No heart attack
Day 56
Citrulline fed
6
6
Water fed
9
4
Citrulline fed
7
10
Water fed
10
4
Procedure
1. Mice were fed citrulline
(once/day for 56 days)
2. Blood was collected at Day 28
and 56. Plasma was separated.
3. Plasma was diluted with 0.25x PBS
Citrulline
4. Diluent was centrifuged
through a filter
Derivatizing
reagent
5. Eluent was aliquotted
and derivatized
0.50
(HPLC column)
Asp - 2.616
2.688
2.409
Gly - 2.451
2.493
2.00
Arg - 2.275
2.317
1.80
2.143
1.982
0.10
His - 1.790
1.828
Ser - 1.857
NH3 - 1.707
0.20
2.982
0.30
AU
7. Data was analyzed for
significant results by
3-way ANOVA
Cit - 2.838
0.40
Glu - 2.908
6. Derivative was analyzed by
amino acid analysis, HPLC
0.00
1.60
2.20
2.40
2.60
2.80
3.0
Results
• The observed plasma amino acid levels were
comparable to published values
• The amino acid levels between males and females
showed no significant difference by T-test
• There were no significant changes in the plasma
concentration of arginine, citrulline, or ornithine
between mice that were gavaged with water versus
mice gavaged with citrulline.
Discussion
• The observed plasma amino acid levels were
comparable to published values
– This suggested our amino acid analysis procedure was
accurate and precise
• Amino acid levels between males and females
showed no significant difference by T-test
– So values from males and females were combined for
the 3-way ANOVA (analysis of variance)
• Our analysis would have detected a change in
plasma amino acid concentration of 30% or
greater.
– Thus, within that limit, there were no significant
changes for arginine, citrulline, or ornithine plasma
concentrations upon citrulline gavage.
PIN
eNOS
iNOS
nNOS
L-arg
ARG1
ARG2
Urea
NO+Citrulline
Aspartate
AS
ASL
Argininosuccinate
Fumarate
Ornithine
ODC
AZI
Putrescine
SPDS
SSAT
AZ
Polyamines
Would increased levels of nitric oxide make it
easier to survive a heart attack?
• Questions to answer
1. Can plasma arginine and citrulline concentrations be
increased by dietary supplementation with
citrulline?
No
2. Do higher blood levels of arginine lead to more nitric
oxide in cardiomyocytes? ???
3. Does the heart show improved recovery from a
myocardial infarction if the animal is provided with
supplemental dietary citrulline
Yes
Q3. Does the heart show improved recovery from a
myocardial infarction if the animal is provided with
supplemental dietary citrulline?
Of the mice who
received a heart
attack…
mice who received
citrulline showed
better fractional
shortening than
mice who received
water
Thorat, S. et al. (2008) Effect of Citrulline Feeding on Extracellular Matrix Remodeling
and Left Ventricular Function in the Mouse Heart Post-Myocardial Infarction (MI)
Conclusion
• There did seem to be an effect of citrulline
feeding
– But this effect was not apparent in the blood
arginine, ornithine, or citrulline concentrations
• If the experiment were to be repeated
– Look for nitric oxide directly
– Look for changes in amino acids in tissues rather
than blood
Acknowledgements
• UW INBRE
• Dr. Mark Stayton, Dept. of Molecular Biology
• Dr. Paul Thomas, Div. of Kinesiology & Health
References
•
Harpster, MH. et al. Earliest changes in the left ventricular transcriptome postmyocardial
infarction. Mamm Genome. 2006;17(7):701-715.
•
Pengxiang S. et al. Obesity-related elevations in plasma leucine are associated with
alterations in enzymes involved in branched-chain amino acid metabolism. Am J Physiol
Endocrinol Metab. 2007; 293(6):E1552-E1563.
•
Schulman, SP. et al. L-Arginine Therapy in Acute Myocardial Infarction, the Vascular
Interaction With Age in Myocardial Infarction (VINTGE MI) Randomized Clinical Trial. JAMA.
2006; 295(1):58-64.
•
Thorat, S. et al. Effect of Citrulline Feeding on Extracellular Matrix Remodeling and Left
Ventricular Function in the Mouse Heart Post-Myocardial Infarction (MI). 2008.