INTERORGAN AMMONIA TRAFFICKING IN HEALTH AND DISEASE

PRESENTOR-DR RAJESH PADHANPRECEPTOR-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 ammoniaUtilization of ammoniaEnzyme involved in ammonia metabolismArterial ammonia levelAmmonia trafficking in healthAmmonia trafficking in diseaseAmmonia 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

• Dietary amino acid• Dietary amine• Intestinal bacteria• Glutamine• Nucleic acid

Utilization of Ammonia• 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• 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

GlutamineTrojan horse

Lacey and Wilmore 1990 Nutr. Rev. 48, 297–309

INTER ORGAN TRAFFICKING IN HEALTHIntestine 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

Small

Intestine (50%) Jejunum 28 % and ileum 22%

Colon (50%)

Portal venous ammonia

Weber et al 1979;Gastroenterology 77

Ammonia metabolism in liver• Excessive dietary nitrogen is either excreted or

converted to a non-toxic form. • Site of detoxification- peri-portal and peri-

venous 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;25:427-

435)• 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

• 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 SHAM(n=8) ALF(n=8)

T= -2 SHAM OPERATION PORTOCAVAL SHUNT

T=0 BLOOD AND URINE COLLECTION

BLOOD AND URINE COLLECTION

HEPATIC ARTERY LIGATION

T=2

T=4 BLOOD AND URINE COLLECTION

BLOOD,URINE AND TISSUE COLLECTIONT=6

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 statusPost pranandial venous ammoniaMental state degradationPortosystemic 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 monitoringUrine 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 disease.

THANK U