Download INTERORGAN AMMONIA TRAFFICKING

INTERORGAN AMMONIA
TRAFFICKING IN HEALTH AND DISEASE
PRESENTOR-DR RAJESH PADHAN
PRECEPTOR-DR S K ACHARYA
Why Ammonia is important ?
• Ammonia is a neurotoxin
• Ammonia is important cause of cerebral
dysfunction in liver failure
• Liver diseases are heterogeneous and manifest
depending on the hepatocyte damage and
hepatocyte reserve.
• Arterial ammonia level depend upon amount of
hepatocyte damage.
• Severity of encephalopathy in ALF depends on
ammonia level
• Ammonia lowering strategies remain primary
therapeutic target for Rx of increased ICP in ALF.
An Egyptian God Amen
To the Greeks Ammon
• Camel urine was collected in a cesspool close to the temple
and it was widely believed that “man and all life rose
spontaneously from a sea of ammonia” camel urine, soot
and sea salt were heated together to form sal ammoniac or
“salt of Ammon”
( Pickett, J., et al. (2000). The American Heritage Dictionary of the English Language)
• Heating of sal ammoniac with alkali resulted in the
production of ammonia gas leading the Swedish chemist T
Bergman to coin the term “ammonia” in 1782
“smelling gas” to revive fainting spells
First published work in 1893 described the physiological
consequences of a portacaval shunt (PCS) in dogs, a surgical
procedure first described by Eck in 1879 (“Eck’s fistula”)
Chief chemist Imperial Institute of
Experimental Medicine in St-Petersburg
Physiologist, St-Petersburg
What are the issues in Ammonia metabolism?
How it exist ?
Sources of ammonia
Utilization of ammonia
Enzyme involved in ammonia metabolism
Arterial ammonia level
Ammonia trafficking in health
Ammonia trafficking in disease
 Ammonia lowering therapies
How it exist in the body ?
• NH3 exists as free form and bound form NH4 +.
• 98% of total ammonia exists as NH4 +
• NH3 is main diffusible form transported across
biological membranes
• Transport is facilitated by constitutive ion channels
and transporters such as the Rhesus proteins and
Aquaporin
Sources of ammonia
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Dietary amino acid
Dietary amine
Intestinal bacteria
Glutamine
Nucleic acid
Utilization of Ammonia
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Liver
Kidney
Muscle
Brain
Production and utilization of Ammonia
Level of Arterial Ammonia
• Healthy volunteers - about 45µM
(Clemmesen ,2000; Gastroenterology 118)
• Chronic liver failure , level is elevated to 60 µM
( Clemmesen ,2000; Gastroenterology 118)
( Plauth et al., 2000; Gut 46 )
• Higher arterial ammonia are documented in ACLF (90–120µ
M) and ALF (150–180µ M)
(Clemmesen et al.,1999 Hepatology 29 )
• Highest concentrations were found with ALF and elevated
intracranial pressure that was unresponsive to conventional
treatment (340µ M)
(Jalan et al., 1999 Lancet 354)
• Portacaval shunted rats showed 2-3 fold increase in ammonia
concentration with normal control rats
( Dejong et al., 1992 Gastroenterology 102)
Hyperammonemia -A main contributor to death
• Arterial NH3 (>124mmol/l) at admission is
predictive of outcome of pts with ALF
(V Bhatia, R Singh, S K Acharya Gut 2006;55:98–104)
• Arterial NH3 > 146 mol/l has been proposed as a
predictor of brain herniation & mortality in pts with
ALF
(Clemmesen et al., Hepatology 1999; 29: 648–653)
• Arterial NH3 concentration, delivery to the brain
and metabolic rate are higher in pts with high
intracranial pressure
(Jalan et al. Lancet 1999;354,
1164–1168)
.
Enzymes involved in Ammonia metabolism
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Glutaminase
Glutamine synthetase
Transamidation reaction
Urea cycle
Urease
Amino oxidase
Relationship between ammonia, glutamate and
glutamine
GLUTAMINE
• Non-toxic, non-essential amino acid
• Highest plasma concentration(50% of the
whole body free amino acid pool)
• Glutamine is the most abundant in protein
• Role -1) fuel for intestinal and other rapidly
dividing cells e.g. immune system
2) regulation of acid/base balance by
amniogenesis
50% of free
AA
Ammonia
donor excretion
of protons in
kidney
Transmitter
glutamate
Glutamine
Trojan
horse
Fuel for gut,
kidney,
immune
system cells
Ammonia
acceptor: Brain:
formation of gln
from glu and NH4
Most
abundant
AA in blood
Lacey and Wilmore 1990 Nutr. Rev. 48, 297–309
INTER ORGAN TRAFFICKING IN HEALTH
Intestine
 Liver
 Kidney
 Muscle
 Brain
Ammonia and glutamine exchange across the gut
• Glutamine is crucial source of energy for SI.
• Intestine takes up glutamine in large quantities from either
blood or intestinal lumen (Adv Enzym 1982, 53:201–237).
• Glutamine is predominantly consumed in jejunum
• Glutaminase - 80 % in small bowel and 20% in large bowel
• High glutaminase activity in small intestine mucosa produce
glutamate and ammonia from glutamine
• Large bowel utilize less glutamine but utilize glucose, short
chain fatty acid and ketones
• Large intestine contribute significantly to portal venous
ammonia concentration by bacterial splitting of urea and
aminoacid
(Gastroenterology,1979:235-240)
Intestinal Ammonia and amino acid production
Contribution of portal ammonia
Portal
venous
ammonia
Colon (50%)
Weber et al 1979;Gastroenterology 77
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Ammonia metabolism in liver
Excessive dietary nitrogen is either excreted or
converted to a non-toxic form.
Site of detoxification- peri-portal and perivenous hepatocytes
Periportal hepatocyte-prominent site for
hepatic urea cycle and glutaminase activity.
Urea cycle convert ammonia to urea
1 mole of urea remove 2 mol of waste nitrogen
Interaction between urea cycle and Krebs cycle
Perivenous hepatocyte
• 7 % of hepatocyte
• Abundant GS convert ammonia to glutamine
• Any ammonia escape periportal hepatocyte
can be scavenged and detoxified by
perivenous hepatocyte
Ammonia and glutamine metabolism in the liver
Kidney and ammonia metabolism
• Kidney contain both glutaminase and glutamine
synthetase enzyme
• Glutamine is the main substrate for renal
ammoniagenesis
• Normal physiological state-kidney excrete only
minor amount of ammonia and 30 % of total
ammonia production is released into urine and
70 % is released in to renal vein.
• During acidosis, total ammoniagenesis is
enhanced and 70 % of this enhanced amount is
excreted in the urine to dispose the acid load.
Role of kidney in ammonia metabolism
Role of kidney in inter organ ammonia exchange due to liver failure
Ammonia and muscle
• Muscle is devoid of an effective urea cycle and relies
exclusively on glutamine production
• GS activity in muscle is low (Metabolism 1976;25:427435)
• Due to large muscle mass, it has great impact on nitrogen
metabolism
• Skeletal muscle glutaminase activity is negligible as
compare to GS activity
• Glucose-alanine cycle- ammonium ion is transported from
muscle cells to the liver in the form of alanine
Skeletal muscle ammonia and aminoacid metabolism
Ammonia and brain
• Normal brain is an organ of ammonia uptake and
glutamine release
• Ammonia readily traverses BBB with positive arterial–
venous differences suggesting net brain ammonia
uptake
• Brain contains appreciable amounts of both glutamine
synthetase and glutaminase
(Cooper and Plum 1987)
• Astrocytes contain most of total brain glutamine
synthetase while neurons contain virtually all brain
glutaminase
(Cooperand Plum 1987).
• Astrocyte GS preferentially takes up ammonia to form
glutamine, which is deaminated to form GABA and
glutamate
Ammonia neurotoxicity
• Impaired bioenergetics and neurotransmission
• Astrocyte swelling-Glutamine synthetase predominant in
astrocyte location and NH3 result in alteration of key astrocyte
protein including glial fibrillary acidic protein ,glutamine and
alanine trasporter
• Oxidative stress-Decrese activity of free radical scavenging
system
• Nitrosative stress-NH3 result in increase concentration of L
tryptophan metabolite including serotonin and quinolnic acid.
• Mitochondrial dysfunction-Ammonia inhibit TCA cycle
• Increased neuro-steroid biosynthesis-Ammonia modulate
expression of PTBR which mediate cholesterol tranport and
biosynthesis of neurosteroid-GABA-A and NMDA receptor
• Direct affect on excitatory and inhibitory receptor function.
(Neurochem Int. 2002;41:109-14)(,prog Neurobiol 2002;67:259-79)
Ammonia and Other organs
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Lung
Heart
Adipose tissue
Immune cell
(LIMITED EVIDENSE ?)
INTERORGAN TRAFFICKING IN DISEASE
Ammonia metabolism in intenstine in Liver failure
• Cirrhosis is associated with 4 folds increase in intestinal PAG
activity in the small bowel (j hepatology 2004:41:49-54)
• In stable cirrhotic patients with TIPS there is net intestinal
ammonia production, which directly correlates with glutamine
uptake (Hepatology 2002;36:1163-1171)
• The kidney plays a major role in the hyperammonia seen after
stimulated or actual GI bleeding in patients with cirrhosis
(Hepatology 2003:37:1277-1285)
• In pigs induction of ALF did not provoke net intenstinal
ammonia production
(Am J Physiol Gastrointest Liver Physiol 200 :291: G373–
81)
Ammonia metabolism in Liver in liver failure
• Hepatocyte loss reduces ammonia detoxification
by reducing the quantity of periportal urea and
perivenous glutamine synthesis
• Portal–systemic shunting further reduces
ammonia detoxification
• With progressive liver injury, despite increases in
periportal glutaminase activity (six-fold) and
ureagenesis increasing amounts of ammonia pass
through to the terminal venules as corresponding
perivenous glutamine synthetic activity is not
increased.
Ammonia metabolism in Muscle in liver failure
• Skeletal muscle ammonia uptake is correlated to
arterial levels in ALF and cirrhosis
• In ALF patients with advanced HE, skeletal muscle
consumes ammonia (100 nmol/100 g/min) with the
stoichiometric release of glutamine (Gastroenterology 2000)
• Hyperammonaemic cirrhotics who underwent TIPS
for gastrointestinal bleeding, skeletal muscle was also
the main site of ammonia removal
(Hepatology 2003)
Ammonia metabolism in kidney in liver failure
• Ammonia excretion is not directly correlated to
plasma levels
(Gastroenterology1960; 39: 420–4)
• Early nineties –Rat experiment showed that in acute
and chronic hyperammonia –reversal of urine
excretion /renal venous ratio from 30/70 to 70/30
(Hepatology 18:890-902)
• Recent study showed in stimulated GIB,increase
ammonia concentration is due to increased renal
amniogenesis
(Hepatology 37:1277-1285)
Ammonia metabolism in Brain in liver failure
• Brain delivery, extraction and uptake of ammonia increases
in ALF and correlates with arterial levels
(J Clin Invest 1955; 34:622–8)
• Ammonia detoxification produces glutamine accumulation
and thus osmotic stress – the ‘ammonia-glutamine-brain
swelling hypothesis
( J Hepatol 2000; 32: 1035–8)
• Glutamine increases
Astrocytes expel myo-inositol and
other weaker osmolytes maintain osmotic equilibrium.
• ALF patients rapid rise in ammonia may outstrip
compensatory mechanisms – Cerebral edema
• In cirrhosis- more gradual increase in plasma ammonia
there is some protection from intracranial hypertension and
brain oedema
Interorgan ammonia metabolism in health and disease
Interorgan ammonia,glutamate and glutamine trafficking in ALF
16 female
Norwegian pigs
Randomised
T= -2
T=0
SHAM(n=8)
ALF(n=8)
SHAM OPERATION
PORTOCAVAL SHUNT
BLOOD AND URINE COLLECTION
HEPATIC ARTERY LIGATION
T=2
BLOOD AND URINE COLLECTION
T=4
BLOOD AND URINE COLLECTION
T=6
BLOOD,URINE AND TISSUE COLLECTION
Results
• ALF pigs develop hyperammonemia and incresed
glutamine level whereas glutamate levels were
decreased.
• PDV contributed to the hyperammonemic state
• Mainly through increased shunting and not as a
result of increased glutamine breakdown.
• kidneys were quantitatively as important as PDV
in systemic ammonia release, whereas muscle
took up ammonia.
• Lungs are able to remove ammonia from the
circulation during the initial stage of ALF.
Ammonia Lowering therapy
Arginine supplementation
• It is a semisynthetic aminoacid
• L -arginine supplementation-allow ammonia
detoxification to urea via arginase.
• No study to evaluate its role in HE
Phenylbutyrate
• Phenyalbutyrate >> phenylacetate
• Phenylacetate+Glutamine=phenyalacetylgluta
mine(remove glutamine)
• Trialed in HE
Sodium benzoate
• Activated by conjugation with CoA and the generated benzoyl CoA is
then conjugated to glycine to form hippurate, which can be
eliminated in the urine
• Ammonia is consumed to replenish the glycine used in the hippurate
synthesis
• initially reported to successfully control episodes of
hyperammonemia in patients born with genetic defects of urea cycle
enzyme
• Elimination of benzoate may induce a depletion of CoA
Gastroenterology 2000; 12: 95-102
Sodium benzoate
• Study –Randomised control trail
• Patients-74 Pts with cirrhosis or portosystemic
anastomosis and hepatic encephalopathy of <7 days
• Treatment –Sodium benzoate (38 ) and lactulose (36 )
• Result-30 patients (80 %) receiving sodium benzoate
and 29 (81 %) receving lactulose recovered
• Conclusion-sodium benzoate is a safe and effective
alternative to lactulose in Acute HE.
(Hepatology 1992;16:138-144)
L-Ornithine L -Aspartate
• LOLA is a compound salt of ornithine and aspartate.
• In the periportal hepatocytes ornithine serves as an
activator of ornithine transcarbamoylase and
carbamoyl phosphate synthetase.
• Ornithine itself acts as a substrate for urea genesis.
• Aspartate and ornithine after conversion to ketoglutarate, also serves as carbon sources for
perivenous glutamine synthesis.
• In the skeletal muscle, LOLA up-regulates glutamine
synthesis by substrate provision for glutamine
synthetase
LOLA in ALF
Efficacy of L-Ornithine L-Aspartate in Acute Liver Failure
(Acharya S K. etal GASTROENTEROLOGY 2009;136:2159–2168 )
LOLA in Rats with acute liver failure
(Hepatology 1999:636-640)
LOLA in cirrhosis and Hepatic encephalopathy
• Study-126 pts with subclinical HE and manifest
HE(grade I and II) -63 Placebo and 63 OA
• Parameter studyNCT-A Performance status
Post pranandial venous ammonia
Mental state degradation
Portosystemic encephalopathy index
(Hepatology 1997;25:1351-1359)
L-0rnithine phenylacetate
BENEFIT –LACK OF REBOUND HYPERAMMONIA
L-0rnithine phenylatate in ALF
• Study -24 pigs-3 groups
8-Sham operated +vehicle
8-ALF +Vehicle
8-ALF + L-Ornithine +phenylbutyrate
• Parameter Arterial and extracellular brain ammonia
ICP monitoring
Urine phenyl acetylglutamine
Results
• Compared with ALF saline pigs, treatment with
OP significantly attenuated concentrations of
arterial ammonia (589.6± 56.7 vs 365.2± 60.4
mol/L (P = 0.002) and extracellular brain
ammonia (P= 0.01).
• ICP - ALF +saline= 18.3± 1.3 mmHg
ALF + OP = 10.3±1.1 mmHg P -0.001)
• Urine phenylacetylglutamine levels increased to
4.9 ± 0.6 mol/L in ALF OP-treated pigs versus
0.50 ±.04mol/L in ALF saline-treated pigs
(P<0.001).
Arterial ammonia concentration over time
(Hepatology 2009:165-174)
L-Ornithine and phenylacetate in Cirrhosis rat
• Model-Bile duct ligation model
• Study-Two parts
 3hr study to determine wheather L-ornithine and
phenylbutyrate were synergistic
 10 day study to determine whether administration of
OP achieved sustained reduction in ammonia
Results (study 1)
Results (study 2)
Survival after treatment with phenylaacetate and
benzoate for urea cycle disorder
• Study-open label, uncontrolled ,non
randomized study in united state from Aug
1980 to march 2005
• Patient-229 patients having urea cycle defect
• Results=over all survival was 84 %
• 96 % survived episodes of hyper ammonia.
• 81 % comatose at admission=survived
(Nejm356:22.2007)
Conclusion
• Liver has key function in nitrogen metabolism
• Ammonia is important in several biochemical
pathway but toxic at elevated level.
• Ammonia is central in mechanism of encephalopathy
in liver failure
• Normally ammonia is present at nontoxic level
• Hyperammonia in chronic liver disease is adapted
increased uptake in muscle but this adaptive
mechanism fells in ALF due to massive rapid
hepatocyte damage leading to sudden increase in
ammonia,thus cerebral edema.
• Ammonia lowering therapy help in chronic liver
THANK U