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2 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.

3 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

4 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

5 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

6 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

7 Sources of ammonia Dietary amino acid Dietary amine Intestinal bacteria Glutamine Nucleic acid

8 Utilization of Ammonia Liver Kidney Muscle Brain

9 Production and utilization of Ammonia

10 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)

11 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).

12 Enzymes involved in Ammonia metabolism Glutaminase Glutamine synthetase Transamidation reaction Urea cycle Urease Amino oxidase

13 Relationship between ammonia, glutamate and glutamine

14 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

15 Glutamine Trojan horse 50% of free AA Transmitter glutamate Ammonia acceptor: Brain: formation of gln from glu and NH4 Most abundant AA in blood Fuel for gut, kidney, immune system cells Ammonia donor excretion of protons in kidney Lacey and Wilmore 1990 Nutr. Rev. 48, 297–309

16 INTER ORGAN TRAFFICKING IN HEALTH  Intestine  Liver  Kidney  Muscle  Brain

17 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)

18 Intestinal Ammonia and amino acid production

19 Contribution of portal ammonia Colon (50%) Portal venous ammonia Weber et al 1979;Gastroenterology 77

20 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

21 Interaction between urea cycle and Krebs cycle

22 Perivenous hepatocyte 7 % of hepatocyte Abundant GS convert ammonia to glutamine Any ammonia escape periportal hepatocyte can be scavenged and detoxified by perivenous hepatocyte

23 Ammonia and glutamine metabolism in the liver

24 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.

25 Role of kidney in ammonia metabolism

26 Role of kidney in inter organ ammonia exchange due to liver failure

27 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

28 Skeletal muscle ammonia and aminoacid metabolism

29 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

30 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)

31 Ammonia and Other organs Lung Heart Adipose tissue Immune cell (LIMITED EVIDENSE ?)


33 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)

34 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.

35 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)

36 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)

37 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

38 Interorgan ammonia metabolism in health and disease


40 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.

41 Ammonia Lowering therapy

42 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

43 Phenylbutyrate Phenyalbutyrate >> phenylacetate Phenylacetate+Glutamine=phenyalacetylgluta mine(remove glutamine) Trialed in HE

44 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

45 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)

46 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

47 LOLA in ALF

48 Efficacy of L-Ornithine L-Aspartate in Acute Liver Failure (Acharya S K. etal GASTROENTEROLOGY 2009;136:2159–2168 )

49 LOLA in Rats with acute liver failure (Hepatology 1999:636-640)

50 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)

51 L-0rnithine phenylacetate BENEFIT –LACK OF REBOUND HYPERAMMONIA

52 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

53 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).

54 Arterial ammonia concentration over time (Hepatology 2009:165-174)

55 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

56 Results (study 1)

57 Results (study 2)

58 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)

59 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.



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