Just Say “NO!” To Ammonia Kelley E. Capocelli, MD Clinical Pathology Conference April 29, 2005

Overview Review of hepatic encephalopathy Review of hepatic encephalopathy Neurotoxins involved with pathogenesis of hepatic encephalopathy Neurotoxins involved with pathogenesis of hepatic encephalopathy Discussion of ammonia Discussion of ammonia Alternatives to ammonia? Alternatives to ammonia?

Hepatic Encephalopathy Mechanisms Accumulation of nitrous metabolites Accumulation of nitrous metabolites Accumulation of toxic substances Accumulation of toxic substances Hypoglycemia Hypoglycemia Brain edema Brain edema False neurotransmitters False neurotransmitters Electrolyte changes Electrolyte changes Acidosis Acidosis

EEG Abnormalities Nonspecific Nonspecific Slow waves of large amplitude Slow waves of large amplitude Bursts of triphasic wave patterns Bursts of triphasic wave patterns Generally read as “consistent with metabolic encephalopathy” because similar patterns are observed in uremia, pulmonary or heart failure, and acid-base disorders Generally read as “consistent with metabolic encephalopathy” because similar patterns are observed in uremia, pulmonary or heart failure, and acid-base disorders Subject to variability in interpretation Subject to variability in interpretation

PET and Proton MR Studies PET PET Shows significantly decreased glucose utilization in the cerebral cortex and concomitant increased utilization in the thalamus, caudate lobe, and cerebellum Shows significantly decreased glucose utilization in the cerebral cortex and concomitant increased utilization in the thalamus, caudate lobe, and cerebellum These findings suggest that hypometabolism in the brains of patients with chronic liver disease could explain the neuropsychiatric abnormalities characteristic of hepatic encephalopathy These findings suggest that hypometabolism in the brains of patients with chronic liver disease could explain the neuropsychiatric abnormalities characteristic of hepatic encephalopathy Proton MR spectroscopy of brains of cirrhotic patients showed depletion of myoinositol (a sign of increased osmolality) and increased glutamine Proton MR spectroscopy of brains of cirrhotic patients showed depletion of myoinositol (a sign of increased osmolality) and increased glutamine

CT and MRI Important in ruling our intracranial lesions when the diagnosis of hepatic encephalopathy is in question Important in ruling our intracranial lesions when the diagnosis of hepatic encephalopathy is in question MRI is able to demonstrates hyperintensity of the globus pallidus on T1-weighted images, a finding that is commonly described in hepatic encephalopathy MRI is able to demonstrates hyperintensity of the globus pallidus on T1-weighted images, a finding that is commonly described in hepatic encephalopathy May be due to manganese accumulation May be due to manganese accumulation

Neurotoxins in Liver Disease Ammonia Ammonia Normal NH 3 metabolism Normal NH 3 metabolism By-product of protein catabolism by gut flora By-product of protein catabolism by gut flora Absorbed by gut and enters portal vein Absorbed by gut and enters portal vein Metabolized in the liver to urea and glutamine (Krebs cycle) Metabolized in the liver to urea and glutamine (Krebs cycle) NH 3 in liver disease NH 3 in liver disease Not cleared by the liver Not cleared by the liver Delivered to the brain by systemic circulation Delivered to the brain by systemic circulation Associated with hepatic encephalopathy Associated with hepatic encephalopathy Not necessarily the cause!

Neurotoxins in Liver Disease False neurotransmitters – amino acids False neurotransmitters – amino acids Patients with cirrhosis have a decreased ratio of branched-chain amino acids (BCAA) to aromatic acids (AAA), from 3.5:1 to 1:1. Patients with cirrhosis have a decreased ratio of branched-chain amino acids (BCAA) to aromatic acids (AAA), from 3.5:1 to 1:1. BCAA include valine, leucine, and isoleucine BCAA include valine, leucine, and isoleucine AAA include phenylalanine, tyrosine, and tryptophan AAA include phenylalanine, tyrosine, and tryptophan The decrease in BCAA is caused predominantly by their extensive use by skeletal muscle The decrease in BCAA is caused predominantly by their extensive use by skeletal muscle

Neurotoxins in Liver Disease False neurotransmitters (cont.) False neurotransmitters (cont.) It has been postulated that the increase in AAA in the CNS may interfere with physiologic neurotransmission by competitively inhibiting “normal” neurotransmitters (i.e., DA, NE) and favoring formation of weak, “false” neurotransmitters (i.e., octopamine) It has been postulated that the increase in AAA in the CNS may interfere with physiologic neurotransmission by competitively inhibiting “normal” neurotransmitters (i.e., DA, NE) and favoring formation of weak, “false” neurotransmitters (i.e., octopamine)

Neurotoxins in Liver Disease This attractive hypothesis raises the possibility that correction of the AAA:BCAA ratio may lead to amelioration of hepatic encephalopathy This attractive hypothesis raises the possibility that correction of the AAA:BCAA ratio may lead to amelioration of hepatic encephalopathy A multitude of clinical trials have failed to prove that changes in the ratio through IV or oral administration of BCAA result in significant improvement of clinical signs or symptoms of this condition A multitude of clinical trials have failed to prove that changes in the ratio through IV or oral administration of BCAA result in significant improvement of clinical signs or symptoms of this condition

Neurotoxins in Liver Disease Accumulation of manganese Accumulation of manganese Observation that more than 80% of patients with cirrhosis in hepatic coma have increased concentrations of manganese Observation that more than 80% of patients with cirrhosis in hepatic coma have increased concentrations of manganese Prolonged exposure to manganese results in extrapyramidal symptoms and abnormal MR images Prolonged exposure to manganese results in extrapyramidal symptoms and abnormal MR images Long-term exposure to manganese also leads to Alzheimer type II astrocytosis Long-term exposure to manganese also leads to Alzheimer type II astrocytosis Direct measurements of basal ganglia manganese levels in autopsy have shown a twofold to sevenfold increase Direct measurements of basal ganglia manganese levels in autopsy have shown a twofold to sevenfold increase Proposed that ammonia and manganese act synergistically Proposed that ammonia and manganese act synergistically

Neurotoxins in Liver Disease Monoamines Monoamines Many of the early neuropsychiatric symptoms of hepatic encephalopathy (e.g. altered sleep patterns) have been attributed to modification of the monoamine neurotransmitter serotonin Many of the early neuropsychiatric symptoms of hepatic encephalopathy (e.g. altered sleep patterns) have been attributed to modification of the monoamine neurotransmitter serotonin A two to fourfold increase in cerebral concentration of the serotonin metabolite 5- hydroxyindoleacetic acid is the most consistent neurochemical finding in HE A two to fourfold increase in cerebral concentration of the serotonin metabolite 5- hydroxyindoleacetic acid is the most consistent neurochemical finding in HE In addition, HE is associated with alterations in the number of 5HT1A and 5HT2 receptors and increased activity of both MAOA and MAOB (enzymes catabolizing 5-HT) In addition, HE is associated with alterations in the number of 5HT1A and 5HT2 receptors and increased activity of both MAOA and MAOB (enzymes catabolizing 5-HT) The findings suggest an increased serotonin turnover rate in HE The findings suggest an increased serotonin turnover rate in HE

Neurotoxins in Liver Disease Histamine Histamine Autopsied brain tissue from cirrhotic patients with HE displayed a higher density and a lower affinity of histamine H1 receptors compared with control human frontal cortex Autopsied brain tissue from cirrhotic patients with HE displayed a higher density and a lower affinity of histamine H1 receptors compared with control human frontal cortex Binding was highest in the parietal and temporal cortices and lowest in the caudate-putamen Binding was highest in the parietal and temporal cortices and lowest in the caudate-putamen

Neurotoxins in Liver Disease Histamine (cont.) Histamine (cont.) A selective increase in H1 receptor density was also observed in parietal and insular cortices of patients with HE A selective increase in H1 receptor density was also observed in parietal and insular cortices of patients with HE A selective up-regulation of brain H1 could contribute to the neuropsychiatric symptoms characteristic of human HE A selective up-regulation of brain H1 could contribute to the neuropsychiatric symptoms characteristic of human HE May be amenable to treatment with selective histamine H1 receptor antagonists May be amenable to treatment with selective histamine H1 receptor antagonists

Neurotoxins in Liver Disease Oxindole Oxindole A tryptophan metabolite formed by gut bacteria (via indol) that can cause sedation, muscle weakness, hypotension, and coma A tryptophan metabolite formed by gut bacteria (via indol) that can cause sedation, muscle weakness, hypotension, and coma Cerebral concentrations of oxindole are increased 200-fold in rats with acute liver failure Cerebral concentrations of oxindole are increased 200-fold in rats with acute liver failure More than a 50-fold increase in the plasma concentration of oxindole has been found in patients with HE More than a 50-fold increase in the plasma concentration of oxindole has been found in patients with HE The mechanism is currently under investigation The mechanism is currently under investigation

The first experiment implicating a nitrogenous substance as a cause of hepatic encephalopathy was performed by Eck The first experiment implicating a nitrogenous substance as a cause of hepatic encephalopathy was performed by Eck Created portal-systemic shunts in healthy dogs and observed that these dogs promptly became comatose after eating meat Created portal-systemic shunts in healthy dogs and observed that these dogs promptly became comatose after eating meat History of Ammonia

Ammonia is the culprit because… The role of ammonia has been postulated on the basis of the following: The role of ammonia has been postulated on the basis of the following: A reproducible increase in blood ammonia levels of patients with cirrhosis A reproducible increase in blood ammonia levels of patients with cirrhosis The development of hepatic coma in patients with advanced liver disease and in experimental animals after ingestion of ammonia The development of hepatic coma in patients with advanced liver disease and in experimental animals after ingestion of ammonia Elevated serum levels in children with genetic abnormalities of urea cycle synthesis, which are associated with neuropsychiatric changes similar to those of patients with hepatic encephalopathy Elevated serum levels in children with genetic abnormalities of urea cycle synthesis, which are associated with neuropsychiatric changes similar to those of patients with hepatic encephalopathy

Role of ammonia (cont.) Role of ammonia (cont.) Increased cerebral metabolism of ammonia as detected by PET and ammonia 13 isotope Increased cerebral metabolism of ammonia as detected by PET and ammonia 13 isotope Increased permeability of the blood- brain barrier to ammonia Increased permeability of the blood- brain barrier to ammonia Chronic elevations of blood ammonia levels causing characteristic changes in astrocytes Chronic elevations of blood ammonia levels causing characteristic changes in astrocytes Ammonia is the culprit because…

Role of ammonia (cont.) Role of ammonia (cont.) The exposure of the brain to millimolar concentrations of ammonia may impair neuron- astrocyte trafficking and lead to Alzheimer type II astrocytosis The exposure of the brain to millimolar concentrations of ammonia may impair neuron- astrocyte trafficking and lead to Alzheimer type II astrocytosis Ammonia inhibits excitatory postsynaptic potentials, thereby depressing overall CNS function Ammonia inhibits excitatory postsynaptic potentials, thereby depressing overall CNS function

Are all of these data true? Not all data are consistent with the ammonia toxicity theory Not all data are consistent with the ammonia toxicity theory There is poor correlation of ammonia with hepatic encephalopathy There is poor correlation of ammonia with hepatic encephalopathy This condition can be present in the absence of elevated ammonia levels This condition can be present in the absence of elevated ammonia levels Approximately 10% of patients with significant encephalopathy have normal serum ammonia levels Approximately 10% of patients with significant encephalopathy have normal serum ammonia levels Many patients with cirrhosis have elevated ammonia levels without evidence of encephalopathy Many patients with cirrhosis have elevated ammonia levels without evidence of encephalopathy

Are all of these data true? Low ammonia concentrations are associated with neuroexcitatory effects Low ammonia concentrations are associated with neuroexcitatory effects Ammonia does not induce that classic EEG changes associated with hepatic encephalopathy when it is administered to patients with cirrhosis Ammonia does not induce that classic EEG changes associated with hepatic encephalopathy when it is administered to patients with cirrhosis

Ammonia as a diagnostic test Serum ammonia levels are often elevated in patients with cirrhosis Serum ammonia levels are often elevated in patients with cirrhosis The serum ammonium test measures both ionized ammonia (ammonium) and un- ionized ammonia The serum ammonium test measures both ionized ammonia (ammonium) and un- ionized ammonia However, only un-ionized ammonia crosses the membranes However, only un-ionized ammonia crosses the membranes In patients with normal serum pH, the ammonia represents only a small fraction of total ammonium concentrations In patients with normal serum pH, the ammonia represents only a small fraction of total ammonium concentrations This factor may be in part responsible for the poor predictability of total ammonium measurement as a diagnostic or prognostic test This factor may be in part responsible for the poor predictability of total ammonium measurement as a diagnostic or prognostic test

Ammonia and Chemistry In 1859, Berthelot described a reaction between ammonia and an alkaline solution of phenol hypochlorite suitable for the determination of ammonia In 1859, Berthelot described a reaction between ammonia and an alkaline solution of phenol hypochlorite suitable for the determination of ammonia Assay was subject to interferences Assay was subject to interferences

Ammonia and Chemistry In 1963, Kirsten et al introduced an enzymatic method for ammonia determination based on the action of glutamate dehydrogenase In 1963, Kirsten et al introduced an enzymatic method for ammonia determination based on the action of glutamate dehydrogenase Although the enzymatic reaction was highly specific and utilized direct evaluation based on the molar absorption of NADH, there were difficulties in stabilizing the end reaction Although the enzymatic reaction was highly specific and utilized direct evaluation based on the molar absorption of NADH, there were difficulties in stabilizing the end reaction

Ammonia and Chemistry Current method is based on Da Fonseca-Wollheim’s modification of the Kirsten reaction Current method is based on Da Fonseca-Wollheim’s modification of the Kirsten reaction Improvements to the original reaction Improvements to the original reaction Addition of ADP to the reaction mixture Addition of ADP to the reaction mixture The use of NADPH in place of NADH to eliminate interference from the reaction of endogenous LDH with endogenous pyruvate The use of NADPH in place of NADH to eliminate interference from the reaction of endogenous LDH with endogenous pyruvate The substitution of plasma for deproteinized supernatant The substitution of plasma for deproteinized supernatant

Ammonia and Chemistry In the reaction catalyzed by glutamate dehydrogenase (GLDH), ammonia reacts with α-ketoglutarate and NADPH to form glutamate and NADP + In the reaction catalyzed by glutamate dehydrogenase (GLDH), ammonia reacts with α-ketoglutarate and NADPH to form glutamate and NADP + GLDH GLDH α-ketoglutarate + NH NADPH -----> L-Glutamate + NADP + + H 2 O The inclusion of ADP in the reaction mixture causes an acceleration of the rate of conversion and stabilizes the GLDH in the indicated pH range The inclusion of ADP in the reaction mixture causes an acceleration of the rate of conversion and stabilizes the GLDH in the indicated pH range

Ammonia and Chemistry The amount of NADPH oxidized during the reaction is equivalent to the amount of ammonia in the specimen and can be measured photometrically by the resulting decrease in absorbance The amount of NADPH oxidized during the reaction is equivalent to the amount of ammonia in the specimen and can be measured photometrically by the resulting decrease in absorbance The decrease in absorbance due to consumption of NADPH is measured kinetically. The decrease in absorbance due to consumption of NADPH is measured kinetically.

Ammonia and Chemistry Reagent Preparation Reagent Preparation R1 Working Solution = Buffered Substrate, ready to use R1 Working Solution = Buffered Substrate, ready to use R2 Working Solution = NADPH/GLDH/Buffered Substrate R2 Working Solution = NADPH/GLDH/Buffered Substrate Step 1: Reconstitute Bottle 2b (GLDH) with 0.5 mL of Bottle 2a with 0.5 mL of Bottle 2a (Buffered Substrate) (Buffered Substrate) Let stand 10 minutes at room temperature, occasionally swirling gently Let stand 10 minutes at room temperature, occasionally swirling gently

Ammonia and Chemistry Reagent Preparation (cont.) Reagent Preparation (cont.) Step 2: Reconstitute one bottle of Bottle 2 Step 2: Reconstitute one bottle of Bottle 2 (NADPH) with 4.5 mL of Bottle 2a (NADPH) with 4.5 mL of Bottle 2a (Buffered Substrate). Mix by gentle (Buffered Substrate). Mix by gentle inversion. inversion. Step 3: Add 150 µL of reconstituted Bottle 2b Step 3: Add 150 µL of reconstituted Bottle 2b (GLDH) to Bottle 2 from Step 2. Mix (GLDH) to Bottle 2 from Step 2. Mix by gentle inversion. Transfer into the by gentle inversion. Transfer into the R2 empty bottle supplied. Remove R2 empty bottle supplied. Remove bubbles. bubbles.

Ammonia and Chemistry Specimen Collection and Preparation Specimen Collection and Preparation The patient should fast for 6 hours and should not clench fist during phlebotomy. The patient should fast for 6 hours and should not clench fist during phlebotomy. Plasma: Use nonhemolyzed EDTA (lavender tube) plasma. Draw the specimen from a stasis-free vein and centrifuge in a stoppered tube as soon as possible. Collect 1.0 mL of blood. Plasma: Use nonhemolyzed EDTA (lavender tube) plasma. Draw the specimen from a stasis-free vein and centrifuge in a stoppered tube as soon as possible. Collect 1.0 mL of blood. Sample: Place the sample on ice and assay immediately. Sample: Place the sample on ice and assay immediately. Keep all tubes, cuvettes, and vials covered to prevent uptake of ammonia from the air Keep all tubes, cuvettes, and vials covered to prevent uptake of ammonia from the air

Ammonia and Chemistry Specimen Handling Specimen Handling The specimen must be collected on ice and rushed to the lab. The specimen must be collected on ice and rushed to the lab. Centrifuge the specimen and immediately separate plasma from cells, stopper tube, and return plasma to cup of ice, or freeze immediately. Centrifuge the specimen and immediately separate plasma from cells, stopper tube, and return plasma to cup of ice, or freeze immediately. NOTE: Do not leave sample, callibrator, or quality control cups open for longer than 15 minutes. Results may be affected. Repeat assays must be performed on freshly poured cups, due to evaporation of ammonia. NOTE: Do not leave sample, callibrator, or quality control cups open for longer than 15 minutes. Results may be affected. Repeat assays must be performed on freshly poured cups, due to evaporation of ammonia.

Ammonia and Chemistry Ammonia is stable in plasma for 3 hours at 4-8ºC in a stoppered tube. If the specimen is not immediately tested, freeze the specimen. Ammonia is stable in plasma for 3 hours at 4-8ºC in a stoppered tube. If the specimen is not immediately tested, freeze the specimen. Reportable Range: µg/dL Reportable Range: µg/dL Expected Values = Adults µg/dL (for venous plasma) Expected Values = Adults µg/dL (for venous plasma) Venous ammonia concentrations may be as much as twice that of arterial Venous ammonia concentrations may be as much as twice that of arterial If the serum result is greater than 450, dilute with water to bring the result into the analytical measuring range and multiply the result by the dilution factor. If the serum result is greater than 450, dilute with water to bring the result into the analytical measuring range and multiply the result by the dilution factor.

Ammonia and Chemistry NOTE: Automatic rerun should not be used for ammonia samples. If a sample result exceeds 1000 µg/dL, a fresh sample must be poured. NOTE: Automatic rerun should not be used for ammonia samples. If a sample result exceeds 1000 µg/dL, a fresh sample must be poured.

Problems with ammonia as a diagnostic test for HE Increased levels of ammonia may also be seen with: Increased levels of ammonia may also be seen with: GI bleeding – blood cells are hemolyzed in the intestines, releasing protein GI bleeding – blood cells are hemolyzed in the intestines, releasing protein Muscular exertion – muscles produce ammonia when active and absorb it when resting Muscular exertion – muscles produce ammonia when active and absorb it when resting Tourniquet use – ammonia levels can be increased in the blood sample collected Tourniquet use – ammonia levels can be increased in the blood sample collected Drugs that can increase ammonia – alcohol, barbiturates, diuretics, and narcotics Drugs that can increase ammonia – alcohol, barbiturates, diuretics, and narcotics Smoking Smoking

Problems with ammonia as a diagnostic test for HE Venous ammonia levels are inconsistent Venous ammonia levels are inconsistent Levels are influenced by fist clenching, use of a tourniquet, and whether the sample was placed on ice Levels are influenced by fist clenching, use of a tourniquet, and whether the sample was placed on ice HE is only directly related to ammonia levels up to about a twofold increase above normal HE is only directly related to ammonia levels up to about a twofold increase above normal Any further increase of ammonia concentration does not contribute to the further evolution of HE Any further increase of ammonia concentration does not contribute to the further evolution of HE

Problems with ammonia as a diagnostic test for HE Arterial ammonia levels tend to be more accurate than venous but are not more useful Arterial ammonia levels tend to be more accurate than venous but are not more useful Serial ammonia measurements are inferior to clinical assessment in gauging improvement or deterioration in a patient under therapy for hepatic encephalopathy Serial ammonia measurements are inferior to clinical assessment in gauging improvement or deterioration in a patient under therapy for hepatic encephalopathy The grade of HE is more closely related to the partial pressure of gaseous ammonia since gaseous ammonia readily enters the brain The grade of HE is more closely related to the partial pressure of gaseous ammonia since gaseous ammonia readily enters the brain Postprandial ammonia levels may be more closely related to the grade of HE than fasting levels Postprandial ammonia levels may be more closely related to the grade of HE than fasting levels In clinical trials, ammonia is often measured after a standard meal (or glutamine load) In clinical trials, ammonia is often measured after a standard meal (or glutamine load)

Why then is ammonia considered an appropriate diagnostic test? Ong et al (2003) prospectively evaluated the correlation between ammonia levels and the severity of HE in 121 patients with cirrhosis Ong et al (2003) prospectively evaluated the correlation between ammonia levels and the severity of HE in 121 patients with cirrhosis Methods Methods Based the diagnosis of HE on clinical criteria Based the diagnosis of HE on clinical criteria Severity of HE based on the West Haven Criteria for grading of mental status Severity of HE based on the West Haven Criteria for grading of mental status

Why then is ammonia considered an appropriate diagnostic test? Methods (cont.) Methods (cont.) Obtained arterial and venous blood samples from each patient Obtained arterial and venous blood samples from each patient Four types of ammonia measurements were analyzed Four types of ammonia measurements were analyzed Arterial and venous total ammonia Arterial and venous total ammonia Arterial and venous partial pressure of ammonia Arterial and venous partial pressure of ammonia Spearman rank correlations (r s ) were calculated Spearman rank correlations (r s ) were calculated

Why then is ammonia considered an appropriate diagnostic test? Results Results 30 (25%) had grade 0 HE 30 (25%) had grade 0 HE 27 (22%) had grade 1 HE 27 (22%) had grade 1 HE 23 (19%) had grade 2 HE 23 (19%) had grade 2 HE 13 (11%) had grade 4 HE 13 (11%) had grade 4 HE

Why then is ammonia considered an appropriate diagnostic test? Results (cont.) Results (cont.) Each of the four measures of ammonia increased with the severity of HE Each of the four measures of ammonia increased with the severity of HE Arterial total ammonia (r s = 0.61, p ≤.001) Arterial total ammonia (r s = 0.61, p ≤.001) Venous total ammonia (r s = 0.56, p ≤.001) Venous total ammonia (r s = 0.56, p ≤.001) Arterial partial pressure of ammonia (r s = 0.55, p ≤.001) Arterial partial pressure of ammonia (r s = 0.55, p ≤.001) Venous partial pressure of ammonia (r s = 0.52, p ≤.001) Venous partial pressure of ammonia (r s = 0.52, p ≤.001)

Why then is ammonia considered an appropriate diagnostic test? Conclusions Conclusions Ammonia levels correlate with the severity of HE. Ammonia levels correlate with the severity of HE. Venous sampling is adequate for ammonia measurement Venous sampling is adequate for ammonia measurement There appears to be no additional advantage of measuring the partial pressure of ammonia compared with total ammonia levels There appears to be no additional advantage of measuring the partial pressure of ammonia compared with total ammonia levels

Things aren’t always as they seem The authors evaluated this clinical controversy in 121 patients The authors evaluated this clinical controversy in 121 patients 41% had documented cirrhosis by liver biopsy 41% had documented cirrhosis by liver biopsy 59% had evidence of portal hypertension 59% had evidence of portal hypertension Alcohol was the cause of cirrhosis in 64% of patients Alcohol was the cause of cirrhosis in 64% of patients 53% of patients were receiving lactulose before hospital admission 53% of patients were receiving lactulose before hospital admission 69% were a Child-Pugh Class C 69% were a Child-Pugh Class C

Things aren’t always as they seem Not stated in their abstract Not stated in their abstract There was substantial overlap in the total ammonia concentrations by grade of HE There was substantial overlap in the total ammonia concentrations by grade of HE At least 1 patient with grade 4 HE had a total ammonia (arterial and venous) <25 mmol/L At least 1 patient with grade 4 HE had a total ammonia (arterial and venous) <25 mmol/L Several patients with grade 0 to 2 had ammonia concentrations >100 mmol/L Several patients with grade 0 to 2 had ammonia concentrations >100 mmol/L 69% of their patients without signs or symptoms of HE had total arterial ammonia concentrations >47 mmol/L, which was the upper limit of normal for their institution’s clinical laboratory 69% of their patients without signs or symptoms of HE had total arterial ammonia concentrations >47 mmol/L, which was the upper limit of normal for their institution’s clinical laboratory

Things aren’t always as they seem Phenomenon due to? Phenomenon due to? The variability in ammonia concentrations observed throughout the day The variability in ammonia concentrations observed throughout the day The delay between elevation of ammonia concentrations and degree of encephalopathy seen in some patients The delay between elevation of ammonia concentrations and degree of encephalopathy seen in some patients Other compounds may be involved in the development of HE Other compounds may be involved in the development of HE The most common precipitating factors for grade 1 or higher HE in their patients were azotemia, infection, GI bleeding, lactulose noncompliance, and constipation The most common precipitating factors for grade 1 or higher HE in their patients were azotemia, infection, GI bleeding, lactulose noncompliance, and constipation

Things aren’t always as they seem Clinical implications of this article Clinical implications of this article A single ammonia concentration, whether venous or arterial, has little utility in the diagnosis of HE A single ammonia concentration, whether venous or arterial, has little utility in the diagnosis of HE The diagnosis of HE should be based on the mental status assessment and clinical course of the patient during their hospitalization The diagnosis of HE should be based on the mental status assessment and clinical course of the patient during their hospitalization

Are there alternative tests? Glutamine Glutamine CSF levels CSF levels Not available at DHMC or Specialty Labs Not available at DHMC or Specialty Labs Available at Biochemical Genetics Laboratory, Dr. Goodman Available at Biochemical Genetics Laboratory, Dr. Goodman 1 or 2 day TAT 1 or 2 day TAT $100 $100 Manganese Manganese Not an available test at either DHMC or Specialty Labs Not an available test at either DHMC or Specialty Labs

Are there alternative tests? Serotonin Serotonin Procedure Procedure Patient should avoid avocado, banana, tomato, plum, walnut, pineapple, eggplant, tobacco, tea, and coffee for 3 days before the test Patient should avoid avocado, banana, tomato, plum, walnut, pineapple, eggplant, tobacco, tea, and coffee for 3 days before the test Requires 4 mL of whole blood EDTA Requires 4 mL of whole blood EDTA Transfer EDTA whole blood to plastic tube, add 4 mg ascorbic acid, mix and freeze within 2 hours of collection Transfer EDTA whole blood to plastic tube, add 4 mg ascorbic acid, mix and freeze within 2 hours of collection Test discontinued at DHMC due to difficulty of preparing specimen Test discontinued at DHMC due to difficulty of preparing specimen Available as send out to Specialty Labs with a 30-day TAT Available as send out to Specialty Labs with a 30-day TAT No test available to test levels in CSF No test available to test levels in CSF

Are there alternative tests? Histamine Histamine Test available to test levels in urine Test available to test levels in urine No test available at DHMC or Specialty Labs to test levels in serum or CSF No test available at DHMC or Specialty Labs to test levels in serum or CSF Oxindole Oxindole No test available at either DHMC or Specialty Labs for either serum or CSF No test available at either DHMC or Specialty Labs for either serum or CSF

How our medicine colleagues should respond A 56 year old male is brought to the hospital by his daughter. She found him unconscious that morning on the sidewalk outside his home. At the hospital he is still unconscious and does not respond to painful stimuli. A 56 year old male is brought to the hospital by his daughter. She found him unconscious that morning on the sidewalk outside his home. At the hospital he is still unconscious and does not respond to painful stimuli. Laboratory results Laboratory results Ammonia level 80 (URL = 65) Ammonia level 80 (URL = 65) AST 90 (URL = 44) AST 90 (URL = 44) Albumin 2.2 (LRL = 3.5) Albumin 2.2 (LRL = 3.5)

The intern says The intern says “Well, I thought this might be hepatic encephalopathy, but given how sick this guy is, I expected a MUCH higher ammonia level!” How our medicine colleagues should respond

The attending responds The attending responds “I am not surprised. While elevated ammonia is associated with hepatic encephalopathy, there is not a correlation between this level and disease severity. The patient has other lab findings suggestive of end-stage liver disease. In the future, please do not waste my time, or that of the laboratory techs and the pathology resident, by ordering such a useless test in an adult. Let’s try lactulose and see how the patient responds!”

References Abou-Assi S, Vlahcevic ZR. Hepatic encephalopathy: metabolic consequence of cirrhosis is often reversible. Postgrad Med 2001;109(2): Abou-Assi S, Vlahcevic ZR. Hepatic encephalopathy: metabolic consequence of cirrhosis is often reversible. Postgrad Med 2001;109(2): Ammonia Technical Procedure, DHMC, Reviewed 1/10/2005 Ammonia Technical Procedure, DHMC, Reviewed 1/10/2005 Blei AT, Cordoba J. Hepatic Encephalopathy. The American Journal of Gastroenterology 2001;96(7): Blei AT, Cordoba J. Hepatic Encephalopathy. The American Journal of Gastroenterology 2001;96(7): Ferenci, P. Clinical manifestations and diagnosis of hepatic encephalopathy UpToDate. Ferenci, P. Clinical manifestations and diagnosis of hepatic encephalopathy UpToDate.

References Ferenci, P. Pathogenesis of hepatic encephalopathy. 2005UpToDate. Ferenci, P. Pathogenesis of hepatic encephalopathy. 2005UpToDate. Ong JP, Aggarwal A, Krieger D, Easley KA, et al. Correlation between ammonia levels and the severity of hepatic encephalopathy. Am J Med 2003;114: Ong JP, Aggarwal A, Krieger D, Easley KA, et al. Correlation between ammonia levels and the severity of hepatic encephalopathy. Am J Med 2003;114: Wolf, DC. Hepatic Encephalopathy. eMedicine.com Wolf, DC. Hepatic Encephalopathy. eMedicine.com