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

Just Say “NO!” To Just Say “NO!” To AmmoniaAmmonia

Kelley E. Capocelli, MDKelley E. Capocelli, MD

Clinical Pathology ConferenceClinical Pathology Conference

April 29, 2005April 29, 2005

OverviewOverview

Review of hepatic Review of hepatic encephalopathyencephalopathy

Neurotoxins involved with Neurotoxins involved with pathogenesis of hepatic pathogenesis of hepatic encephalopathyencephalopathy

Discussion of ammoniaDiscussion of ammonia Alternatives to ammonia?Alternatives to ammonia?

Hepatic Encephalopathy Hepatic Encephalopathy MechanismsMechanisms

Accumulation of nitrous metabolitesAccumulation of nitrous metabolites Accumulation of toxic substances Accumulation of toxic substances HypoglycemiaHypoglycemia Brain edemaBrain edema False neurotransmittersFalse neurotransmitters Electrolyte changesElectrolyte changes AcidosisAcidosis

EEG AbnormalitiesEEG Abnormalities

Nonspecific Nonspecific Slow waves of large amplitude Slow waves of large amplitude Bursts of triphasic wave patternsBursts of triphasic wave patterns

Generally read as “consistent with Generally read as “consistent with metabolic encephalopathy” because metabolic encephalopathy” because similar patterns are observed in uremia, similar patterns are observed in uremia, pulmonary or heart failure, and acid-base pulmonary or heart failure, and acid-base disordersdisorders Subject to variability in interpretationSubject to variability in interpretation

PET and Proton MR PET and Proton MR StudiesStudies

PETPET Shows significantly decreased glucose Shows significantly decreased glucose

utilization in the cerebral cortex and utilization in the cerebral cortex and concomitant increased utilization in the concomitant increased utilization in the thalamus, caudate lobe, and cerebellumthalamus, caudate lobe, and cerebellum

These findings suggest that hypometabolism in These findings suggest that hypometabolism in the brains of patients with chronic liver disease the brains of patients with chronic liver disease could explain the neuropsychiatric could explain the neuropsychiatric abnormalities characteristic of hepatic abnormalities characteristic of hepatic encephalopathyencephalopathy

Proton MR spectroscopy of brains of Proton MR spectroscopy of brains of cirrhotic patients showed depletion of cirrhotic patients showed depletion of myoinositol (a sign of increased myoinositol (a sign of increased osmolality) and increased glutamineosmolality) and increased glutamine

CT and MRICT and MRI

Important in ruling our intracranial lesions Important in ruling our intracranial lesions when the diagnosis of hepatic when the diagnosis of hepatic encephalopathy is in questionencephalopathy is in question

MRI is able to demonstrates hyperintensity MRI is able to demonstrates hyperintensity of the globus pallidus on T1-weighted of the globus pallidus on T1-weighted images, a finding that is commonly images, a finding that is commonly described in hepatic encephalopathydescribed in hepatic encephalopathy May be due to manganese accumulationMay be due to manganese accumulation

Neurotoxins in Liver Neurotoxins in Liver DiseaseDisease

AmmoniaAmmonia Normal NHNormal NH33 metabolism metabolism

By-product of protein catabolism by gut floraBy-product of protein catabolism by gut flora Absorbed by gut and enters portal veinAbsorbed by gut and enters portal vein Metabolized in the liver to urea and Metabolized in the liver to urea and

glutamine (Krebs cycle)glutamine (Krebs cycle) NHNH3 3 in liver diseasein liver disease

Not cleared by the liverNot cleared by the liver Delivered to the brain by systemic Delivered to the brain by systemic

circulationcirculation Associated with hepatic encepAssociated with hepatic encephhalopathyalopathy

Not necessarily the cause!Not necessarily the cause!


False neurotransmitters – amino False neurotransmitters – amino acidsacids Patients with cirrhosis have a decreased Patients with cirrhosis have a decreased

ratio of branched-chain amino acids ratio of branched-chain amino acids (BCAA) to aromatic acids (AAA), from (BCAA) to aromatic acids (AAA), from 3.5:1 to 1:1.3.5:1 to 1:1. BCAA include valine, leucine, and isoleucineBCAA include valine, leucine, and isoleucine AAA include phenylalanine, tyrosine, and AAA include phenylalanine, tyrosine, and

tryptophantryptophan The decrease in BCAA is caused The decrease in BCAA is caused

predominantly by their extensive use by predominantly by their extensive use by skeletal muscleskeletal muscle


False neurotransmitters (cont.)False neurotransmitters (cont.) It has been postulated that the increase It has been postulated that the increase

in AAA in the CNS may interfere with in AAA in the CNS may interfere with physiologic neurotransmission by physiologic neurotransmission by competitively inhibiting “normal” competitively inhibiting “normal” neurotransmitters (i.e., DA, NE) and neurotransmitters (i.e., DA, NE) and favoring formation of weak, “false” favoring formation of weak, “false” neurotransmitters (i.e., octopamine)neurotransmitters (i.e., octopamine)


This attractive hypothesis raises This attractive hypothesis raises the possibility that correction of the the possibility that correction of the AAA:BCAA ratio may lead to AAA:BCAA ratio may lead to amelioration of hepatic amelioration of hepatic encephalopathyencephalopathy A multitude of clinical trials have failed to A multitude of clinical trials have failed to

prove that changes in the ratio through IV or prove that changes in the ratio through IV or oral administration of BCAA result in oral administration of BCAA result in significant improvement of clinical signs or significant improvement of clinical signs or symptoms of this condition symptoms of this condition


Accumulation of manganeseAccumulation of manganese Observation that more than 80% of patients Observation that more than 80% of patients

with cirrhosis in hepatic coma have increased with cirrhosis in hepatic coma have increased concentrations of manganeseconcentrations of manganese

Prolonged exposure to manganese results in Prolonged exposure to manganese results in extrapyramidal symptoms and abnormal MR extrapyramidal symptoms and abnormal MR imagesimages

Long-term exposure to manganese also leads Long-term exposure to manganese also leads to Alzheimer type II astrocytosisto Alzheimer type II astrocytosis

Direct measurements of basal ganglia Direct measurements of basal ganglia manganese levels in autopsy have shown a manganese levels in autopsy have shown a twofold to sevenfold increasetwofold to sevenfold increase

Proposed that ammonia and manganese act Proposed that ammonia and manganese act synergisticallysynergistically


MonoaminesMonoamines Many of the early neuropsychiatric symptoms Many of the early neuropsychiatric symptoms

of hepatic encephalopathy (e.g. altered sleep of hepatic encephalopathy (e.g. altered sleep patterns) have been attributed to modification patterns) have been attributed to modification of the monoamine neurotransmitter serotoninof the monoamine neurotransmitter serotonin

A two to fourfold increase in cerebral concentration of A two to fourfold increase in cerebral concentration of the serotonin metabolite 5-hydroxyindoleacetic acid the serotonin metabolite 5-hydroxyindoleacetic acid is the most consistent neurochemical finding in HEis the most consistent neurochemical finding in HE

In addition, HE is associated with alterations in the In addition, HE is associated with alterations in the number of 5HT1A and 5HT2 receptors and increased number of 5HT1A and 5HT2 receptors and increased activity of both MAOA and MAOB (enzymes activity of both MAOA and MAOB (enzymes catabolizing 5-HT)catabolizing 5-HT)

The findings suggest an increased serotonin turnover The findings suggest an increased serotonin turnover rate in HErate in HE


HistamineHistamine Autopsied brain tissue from cirrhotic Autopsied brain tissue from cirrhotic

patients with HE displayed a higher patients with HE displayed a higher density and a lower affinity of histamine density and a lower affinity of histamine H1 receptors compared with control H1 receptors compared with control human frontal cortexhuman frontal cortex

Binding was highest in the parietal and Binding was highest in the parietal and temporal cortices and lowest in the temporal cortices and lowest in the caudate-putamencaudate-putamen


Histamine (cont.)Histamine (cont.) A selective increase in H1 receptor A selective increase in H1 receptor

density was also observed in parietal density was also observed in parietal and insular cortices of patients with HEand insular cortices of patients with HE

A selective up-regulation of brain H1 A selective up-regulation of brain H1 could contribute to the neuropsychiatric could contribute to the neuropsychiatric symptoms characteristic of human HEsymptoms characteristic of human HE May be amenable to treatment with May be amenable to treatment with

selective histamine H1 receptor antagonistsselective histamine H1 receptor antagonists


OxindoleOxindole A tryptophan metabolite formed by gut A tryptophan metabolite formed by gut

bacteria (via indol) that can cause sedation, bacteria (via indol) that can cause sedation, muscle weakness, hypotension, and comamuscle weakness, hypotension, and coma

Cerebral concentrations of oxindole are Cerebral concentrations of oxindole are increased 200-fold in rats with acute liver increased 200-fold in rats with acute liver failurefailure

More than a 50-fold increase in the plasma More than a 50-fold increase in the plasma concentration of oxindole has been found in concentration of oxindole has been found in patients with HEpatients with HE

The mechanism is currently under investigationThe mechanism is currently under investigation

The first experiment implicating a The first experiment implicating a nitrogenous substance as a cause of nitrogenous substance as a cause of hepatic encephalopathy was hepatic encephalopathy was performed by Eckperformed by Eck Created portal-systemic shunts in Created portal-systemic shunts in

healthy dogs and observed that these healthy dogs and observed that these dogs promptly became comatose after dogs promptly became comatose after eating meateating meat

History of AmmoniaHistory of Ammonia

Ammonia is the culprit Ammonia is the culprit because…because…

The role of ammonia has been postulated The role of ammonia has been postulated on the basis of the following:on the basis of the following: A reproducible increase in blood ammonia A reproducible increase in blood ammonia

levels of patients with cirrhosislevels of patients with cirrhosis The development of hepatic coma in patients The development of hepatic coma in patients

with advanced liver disease and in with advanced liver disease and in experimental animals after ingestion of experimental animals after ingestion of ammoniaammonia

Elevated serum levels in children with genetic Elevated serum levels in children with genetic abnormalities of urea cycle synthesis, which abnormalities of urea cycle synthesis, which are associated with neuropsychiatric changes are associated with neuropsychiatric changes similar to those of patients with hepatic similar to those of patients with hepatic encephalopathyencephalopathy

Role of ammonia (cont.)Role of ammonia (cont.) Increased cerebral metabolism of Increased cerebral metabolism of

ammonia as detected by PET and ammonia as detected by PET and ammonia 13 isotopeammonia 13 isotope

Increased permeability of the blood-Increased permeability of the blood-brain barrier to ammoniabrain barrier to ammonia

Chronic elevations of blood ammonia Chronic elevations of blood ammonia levels causing characteristic changes in levels causing characteristic changes in astrocytesastrocytes



Role of ammonia (cont.)Role of ammonia (cont.) The exposure of the brain to millimolar The exposure of the brain to millimolar

concentrations of ammonia may impair concentrations of ammonia may impair neuron-astrocyte trafficking and lead to neuron-astrocyte trafficking and lead to Alzheimer type II astrocytosisAlzheimer type II astrocytosis

Ammonia inhibits excitatory Ammonia inhibits excitatory postsynaptic potentials, thereby postsynaptic potentials, thereby depressing overall CNS functiondepressing overall CNS function

Are all of these data Are all of these data true?true?

Not all data are consistent with the Not all data are consistent with the ammonia toxicity theoryammonia toxicity theory There is poor correlation of ammonia There is poor correlation of ammonia

with hepatic encephalopathywith hepatic encephalopathy This condition can be present in the absence This condition can be present in the absence

of elevated ammonia levelsof elevated ammonia levels Approximately 10% of patients with significant Approximately 10% of patients with significant

encephalopathy have normal serum ammonia encephalopathy have normal serum ammonia levelslevels

Many patients with cirrhosis have elevated Many patients with cirrhosis have elevated ammonia levels without evidence of ammonia levels without evidence of encephalopathyencephalopathy

Are all of these data Are all of these data true?true?

Low ammonia concentrations are Low ammonia concentrations are associated with neuroexcitatory effectsassociated with neuroexcitatory effects

Ammonia does not induce that classic Ammonia does not induce that classic EEG changes associated with hepatic EEG changes associated with hepatic encephalopathy when it is administered encephalopathy when it is administered to patients with cirrhosisto patients with cirrhosis

Ammonia as a diagnostic Ammonia as a diagnostic testtest

Serum ammonia levels are often elevated Serum ammonia levels are often elevated in patients with cirrhosisin patients with cirrhosis The serum ammonium test measures both The serum ammonium test measures both

ionized ammonia (ammonium) and un-ionized ionized ammonia (ammonium) and un-ionized ammoniaammonia

However, only un-ionized ammonia crosses the However, only un-ionized ammonia crosses the membranes membranes

In patients with normal serum pH, the ammonia In patients with normal serum pH, the ammonia represents only a small fraction of total ammonium represents only a small fraction of total ammonium concentrationsconcentrations

This factor may be in part responsible for the poor This factor may be in part responsible for the poor predictability of total ammonium measurement as a predictability of total ammonium measurement as a diagnostic or prognostic testdiagnostic or prognostic test

Ammonia and ChemistryAmmonia and Chemistry

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


In 1963, Kirsten et al introduced an In 1963, Kirsten et al introduced an enzymatic method for ammonia enzymatic method for ammonia determination based on the action of determination based on the action of glutamate dehydrogenaseglutamate dehydrogenase Although the enzymatic reaction was Although the enzymatic reaction was

highly specific and utilized direct highly specific and utilized direct evaluation based on the molar evaluation based on the molar absorption of NADH, there were absorption of NADH, there were difficulties in stabilizing the end reactiondifficulties in stabilizing the end reaction


Current method is based on Da Fonseca-Current method is based on Da Fonseca-Wollheim’s modification of the Kirsten Wollheim’s modification of the Kirsten reactionreaction Improvements to the original reactionImprovements to the original reaction

Addition of ADP to the reaction mixtureAddition of ADP to the reaction mixture The use of NADPH in place of NADH to The use of NADPH in place of NADH to

eliminate interference from the reaction of eliminate interference from the reaction of endogenous LDH with endogenous pyruvateendogenous LDH with endogenous pyruvate

The substitution of plasma for deproteinized The substitution of plasma for deproteinized supernatantsupernatant


In the reaction catalyzed by glutamate In the reaction catalyzed by glutamate dehydrogenase (GLDH), ammonia reacts dehydrogenase (GLDH), ammonia reacts with with αα-ketoglutarate and NADPH to form -ketoglutarate and NADPH to form glutamate and NADPglutamate and NADP++

GLDHGLDH

αα-ketoglutarate + NH-ketoglutarate + NH44++ + NADPH -----> L-Glutamate + NADP + NADPH -----> L-Glutamate + NADP++ + H + H22OO

The inclusion of ADP in the reaction The inclusion of ADP in the reaction mixture causes an acceleration of the rate mixture causes an acceleration of the rate of conversion and stabilizes the GLDH in of conversion and stabilizes the GLDH in the indicated pH rangethe indicated pH range


The amount of NADPH oxidized The amount of NADPH oxidized during the reaction is equivalent to during the reaction is equivalent to the amount of ammonia in the the amount of ammonia in the specimen and can be measured specimen and can be measured photometrically by the resulting photometrically by the resulting decrease in absorbancedecrease in absorbance

The decrease in absorbance due to The decrease in absorbance due to consumption of NADPH is measured consumption of NADPH is measured kinetically.kinetically.


Reagent PreparationReagent Preparation R1 Working Solution = Buffered Substrate, R1 Working Solution = Buffered Substrate,

ready to useready to use R2 Working Solution = R2 Working Solution =

NADPH/GLDH/Buffered SubstrateNADPH/GLDH/Buffered Substrate

Step 1:Step 1: Reconstitute Bottle 2b (GLDH) 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 Let stand 10 minutes at room temperature, occasionally swirling gentlytemperature, occasionally swirling gently

Ammonia and ChemistryAmmonia and Chemistry Reagent Preparation (cont.)Reagent Preparation (cont.)

Step 2: Step 2: Reconstitute one bottle of Bottle 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:Step 3: Add 150 µL of reconstituted Bottle 2b 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 ChemistryAmmonia and Chemistry Specimen Collection and PreparationSpecimen Collection and Preparation

The patient should fast for 6 hours and should The patient should fast for 6 hours and should not clench fist during phlebotomy.not clench fist during phlebotomy.

Plasma:Plasma: Use nonhemolyzed EDTA (lavender Use nonhemolyzed EDTA (lavender tube) plasma. Draw the specimen from a tube) plasma. Draw the specimen from a stasis-free vein and centrifuge in a stoppered stasis-free vein and centrifuge in a stoppered tube as soon as possible. Collect 1.0 mL of tube as soon as possible. Collect 1.0 mL of blood.blood.

Sample:Sample: Place the sample on ice and assay Place the sample on ice and assay immediately. immediately.

Keep all tubes, cuvettes, and vials covered Keep all tubes, cuvettes, and vials covered to prevent uptake of ammonia from the airto prevent uptake of ammonia from the air

Ammonia and ChemistryAmmonia and Chemistry Specimen HandlingSpecimen Handling

The specimen must be collected on ice and The specimen must be collected on ice and rushed to the lab. rushed to the lab.

Centrifuge the specimen and immediately Centrifuge the specimen and immediately separate plasma from cells, stopper tube, and separate plasma from cells, stopper tube, and return plasma to cup of ice, or freeze return plasma to cup of ice, or freeze immediately.immediately.

NOTE:NOTE: Do not leave sample, callibrator, Do not leave sample, callibrator, or quality control cups open for longer or quality control cups open for longer than 15 minutes. Results may be affected. than 15 minutes. Results may be affected. Repeat assays must be performed on Repeat assays must be performed on freshly poured cups, due to evaporation of freshly poured cups, due to evaporation of ammonia.ammonia.


Ammonia is stable in plasma for 3 hours at Ammonia is stable in plasma for 3 hours at 4-8ºC in a stoppered tube. If the specimen 4-8ºC in a stoppered tube. If the specimen is not immediately tested, freeze the is not immediately tested, freeze the specimen.specimen.

Reportable Range:Reportable Range: 10-450µg/dL 10-450µg/dL Expected Values = Expected Values = Adults 17-80 µg/dL (for Adults 17-80 µg/dL (for

venous plasma)venous plasma) Venous ammonia concentrations may be as much as Venous ammonia concentrations may be as much as

twice that of arterialtwice that of arterial If the serum result is greater than 450, dilute with If the serum result is greater than 450, dilute with

water to bring the result into the analytical water to bring the result into the analytical measuring range and multiply the result by the measuring range and multiply the result by the dilution factor.dilution factor.


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

Problems with ammonia as Problems with ammonia as a diagnostic test for HEa diagnostic test for HE

Increased levels of ammonia may also be Increased levels of ammonia may also be seen with:seen with: GI bleeding – blood cells are hemolyzed in the GI bleeding – blood cells are hemolyzed in the

intestines, releasing proteinintestines, releasing protein Muscular exertion – muscles produce ammonia Muscular exertion – muscles produce ammonia

when active and absorb it when restingwhen active and absorb it when resting Tourniquet use – ammonia levels can be Tourniquet use – ammonia levels can be

increased in the blood sample collectedincreased in the blood sample collected Drugs that can increase ammonia – alcohol, Drugs that can increase ammonia – alcohol,

barbiturates, diuretics, and narcoticsbarbiturates, diuretics, and narcotics SmokingSmoking


Venous ammonia levels are Venous ammonia levels are inconsistentinconsistent Levels are influenced by fist clenching, Levels are influenced by fist clenching,

use of a tourniquet, and whether the use of a tourniquet, and whether the sample was placed on icesample was placed on ice

HE is only directly related to ammonia HE is only directly related to ammonia levels up to about a twofold increase levels up to about a twofold increase above normalabove normal Any further increase of ammonia Any further increase of ammonia

concentration does not contribute to the concentration does not contribute to the further evolution of HEfurther evolution of HE


Arterial ammonia levels tend to be more accurate Arterial ammonia levels tend to be more accurate than venous but are not more usefulthan venous but are not more useful Serial ammonia measurements are inferior to clinical Serial ammonia measurements are inferior to clinical

assessment in gauging improvement or deterioration in assessment in gauging improvement or deterioration in a patient under therapy for hepatic encephalopathya patient under therapy for hepatic encephalopathy

The grade of HE is more closely related to the The grade of HE is more closely related to the partial pressure of gaseous ammonia since partial pressure of gaseous ammonia since gaseous ammonia readily enters the braingaseous ammonia readily enters the brain

Postprandial ammonia levels may be more closely Postprandial ammonia levels may be more closely related to the grade of HE than fasting levelsrelated to the grade of HE than fasting levels In clinical trials, ammonia is often measured after a In clinical trials, ammonia is often measured after a

standard meal (or glutamine load)standard meal (or glutamine load)

Why then is ammonia Why then is ammonia considered an appropriate considered an appropriate

diagnostic test?diagnostic test? Ong et al (2003) prospectively Ong et al (2003) prospectively

evaluated the correlation between evaluated the correlation between ammonia levels and the severity of ammonia levels and the severity of HE in 121 patients with cirrhosisHE in 121 patients with cirrhosis

MethodsMethods Based the diagnosis of HE on clinical Based the diagnosis of HE on clinical

criteriacriteria Severity of HE based on the West Haven Severity of HE based on the West Haven

Criteria for grading of mental statusCriteria for grading of mental status


diagnostic test?diagnostic test? MethodsMethods (cont.) (cont.)

Obtained arterial and venous blood Obtained arterial and venous blood samples from each patientsamples from each patient Four types of ammonia measurements Four types of ammonia measurements

were analyzedwere analyzed Arterial and venous total ammoniaArterial and venous total ammonia Arterial and venous partial pressure of Arterial and venous partial pressure of

ammoniaammonia

Spearman rank correlations (rSpearman rank correlations (rss) ) were calculatedwere calculated


diagnostic test?diagnostic test? ResultsResults

30 (25%) had grade 0 HE30 (25%) had grade 0 HE27 (22%) had grade 1 HE27 (22%) had grade 1 HE23 (19%) had grade 2 HE23 (19%) had grade 2 HE13 (11%) had grade 4 HE13 (11%) had grade 4 HE


diagnostic test?diagnostic test? Results (cont.)Results (cont.)

Each of the four measures of ammonia Each of the four measures of ammonia increased with the severity of HEincreased with the severity of HE Arterial total ammonia (rArterial total ammonia (rss = 0.61, p ≤ .001) = 0.61, p ≤ .001)

Venous total ammonia (rVenous total ammonia (rss = 0.56, p ≤ .001) = 0.56, p ≤ .001)

Arterial partial pressure of ammonia (rArterial partial pressure of ammonia (rss = = 0.55, p ≤ .001)0.55, p ≤ .001)

Venous partial pressure of ammonia (rVenous partial pressure of ammonia (rss = = 0.52, p ≤ .001)0.52, p ≤ .001)


diagnostic test?diagnostic test? ConclusionsConclusions

Ammonia levels correlate with the Ammonia levels correlate with the severity of HE.severity of HE.

Venous sampling is adequate for Venous sampling is adequate for ammonia measurementammonia measurement

There appears to be no additional There appears to be no additional advantage of measuring the partial advantage of measuring the partial pressure of ammonia compared with pressure of ammonia compared with total ammonia levelstotal ammonia levels

Things aren’t always as Things aren’t always as they seemthey seem

The authors evaluated this clinical The authors evaluated this clinical controversy in 121 patientscontroversy in 121 patients 41% had documented cirrhosis by liver 41% had documented cirrhosis by liver

biopsybiopsy 59% had evidence of portal 59% had evidence of portal

hypertensionhypertension Alcohol was the cause of cirrhosis in Alcohol was the cause of cirrhosis in

64% of patients64% of patients 53% of patients were receiving lactulose 53% of patients were receiving lactulose

before hospital admissionbefore hospital admission 69% were a Child-Pugh Class C69% were a Child-Pugh Class C


Not stated in their abstractNot stated in their abstract There was substantial overlap in the total There was substantial overlap in the total

ammonia concentrations by grade of HEammonia concentrations by grade of HE At least 1 patient with grade 4 HE had a total At least 1 patient with grade 4 HE had a total

ammonia (arterial and venous) <25 mmol/Lammonia (arterial and venous) <25 mmol/L Several patients with grade 0 to 2 had ammonia Several patients with grade 0 to 2 had ammonia

concentrations >100 mmol/Lconcentrations >100 mmol/L 69% of their patients without signs or symptoms of 69% of their patients without signs or symptoms of

HE had total arterial ammonia concentrations >47 HE had total arterial ammonia concentrations >47 mmol/L, which was the upper limit of normal for their mmol/L, which was the upper limit of normal for their institution’s clinical laboratoryinstitution’s clinical laboratory


Phenomenon due to?Phenomenon due to? The variability in ammonia concentrations The variability in ammonia concentrations

observed throughout the dayobserved throughout the day The delay between elevation of ammonia The delay between elevation of ammonia

concentrations and degree of encephalopathy concentrations and degree of encephalopathy seen in some patientsseen in some patients

Other compounds may be involved in the Other compounds may be involved in the development of HEdevelopment of HE

The most common precipitating factors for The most common precipitating factors for grade 1 or higher HE in their patients were grade 1 or higher HE in their patients were azotemia, infection, GI bleeding, lactulose azotemia, infection, GI bleeding, lactulose noncompliance, and constipationnoncompliance, and constipation


Clinical implications of this articleClinical implications of this article A single ammonia concentration, A single ammonia concentration,

whether venous or arterial, has little whether venous or arterial, has little utility in the diagnosis of HEutility in the diagnosis of HE

The diagnosis of HE should be based on The diagnosis of HE should be based on the mental status assessment and the mental status assessment and clinical course of the patient during their clinical course of the patient during their hospitalizationhospitalization

Are there alternative Are there alternative tests?tests?

GlutamineGlutamine CSF levelsCSF levels

Not available at DHMC or Specialty LabsNot available at DHMC or Specialty Labs Available at Biochemical Genetics Available at Biochemical Genetics

Laboratory, Dr. Goodman Laboratory, Dr. Goodman 1 or 2 1 or 2 dayday TAT TAT $100$100

ManganeseManganese Not an available test at either DHMC or Not an available test at either DHMC or

Specialty LabsSpecialty Labs


SerotoninSerotonin ProcedureProcedure

Patient should avoid avocado, banana, tomato, plum, Patient should avoid avocado, banana, tomato, plum, walnut, pineapple, eggplant, tobacco, tea, and coffee walnut, pineapple, eggplant, tobacco, tea, and coffee for 3 days before the testfor 3 days before the test

Requires 4 mL of whole blood EDTARequires 4 mL of whole blood EDTA Transfer EDTA whole blood to plastic tube, add 4 mg Transfer EDTA whole blood to plastic tube, add 4 mg

ascorbic acid, mix and freeze within 2 hours of ascorbic acid, mix and freeze within 2 hours of collectioncollection

Test discontinued at DHMC due to difficulty of Test discontinued at DHMC due to difficulty of preparing specimenpreparing specimen

Available as send out to Specialty Labs with a Available as send out to Specialty Labs with a 30-day TAT30-day TAT

No test available to test levels in CSFNo test available to test levels in CSF


HistamineHistamine Test available to test levels in urineTest available to test levels in urine No test available at DHMC or Specialty No test available at DHMC or Specialty

Labs to test levels in serum or CSFLabs to test levels in serum or CSF OxindoleOxindole

No test available at either DHMC or No test available at either DHMC or Specialty Labs for either serum or CSFSpecialty Labs for either serum or CSF

How our medicine How our medicine colleagues should respondcolleagues should respond

A 56 year old male is brought to the A 56 year old male is brought to the hospital by his daughter. She found him hospital by his daughter. She found him unconscious that morning on the sidewalk unconscious that morning on the sidewalk outside his home. At the hospital he is still outside his home. At the hospital he is still unconscious and does not respond to unconscious and does not respond to painful stimuli.painful stimuli. Laboratory resultsLaboratory 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 saysThe intern says““Well, I thought this might be Well, I thought this might be hepatic encephalopathy, but hepatic encephalopathy, but given how sick this guy is, I given how sick this guy is, I expected a MUCH higher expected a MUCH higher ammonia level!”ammonia level!”



The attending respondsThe attending responds

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

ReferencesReferences Abou-Assi S, Vlahcevic ZR. Hepatic Abou-Assi S, Vlahcevic ZR. Hepatic

encephalopathy: metabolic consequence encephalopathy: metabolic consequence of cirrhosis is often reversible. of cirrhosis is often reversible. Postgrad Postgrad MedMed 2001;109(2): 52-70. 2001;109(2): 52-70.

Ammonia Technical Procedure, DHMC, Ammonia Technical Procedure, DHMC, Reviewed 1/10/2005Reviewed 1/10/2005

Blei AT, Cordoba J. Hepatic Blei AT, Cordoba J. Hepatic Encephalopathy. Encephalopathy. The American Journal of The American Journal of Gastroenterology Gastroenterology 2001;96(7): 1968-1975.2001;96(7): 1968-1975.

Ferenci, P. Clinical manifestations and Ferenci, P. Clinical manifestations and diagnosis of hepatic encephalopathy. diagnosis of hepatic encephalopathy. 2005 2005 UpToDate.UpToDate.

ReferencesReferences

Ferenci, P. Pathogenesis of hepatic Ferenci, P. Pathogenesis of hepatic encephalopathy. 2005encephalopathy. 2005UpToDate.UpToDate.

Ong JP, Aggarwal A, Krieger D, Ong JP, Aggarwal A, Krieger D, Easley KA, et al. Correlation Easley KA, et al. Correlation between ammonia levels and the between ammonia levels and the severity of hepatic encephalopathy. severity of hepatic encephalopathy. Am J Med Am J Med 2003;114:188-193.2003;114:188-193.

Wolf, DC. Hepatic Encephalopathy.Wolf, DC. Hepatic Encephalopathy. eMedicine.com eMedicine.com

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