M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 1 (2 0 1 2) 1 49 5 1 5 11

Available online at www.sciencedirect.com

Metabolism
www.metabolismjournal.com

Reviews

Ammonium metabolism in humans

Maria M. Adeva a,, Gema Souto b , Natalia Blanco c , Cristbal Donapetry a

a
Hospital General Juan Cardona
b
Clinical Center of the National Institutes of Health
c
United Surgical Partners. La Corua

A R T I C LE I N FO AB S T R A C T

Article history: Free ammonium ions are produced and consumed during cell metabolism. Glutamine
Received 23 March 2012 synthetase utilizes free ammonium ions to produce glutamine in the cytosol whereas
Accepted 16 July 2012 glutaminase and glutamate dehydrogenase generate free ammonium ions in the
mitochondria from glutamine and glutamate, respectively. Ammonia and bicarbonate are
Keywords: condensed in the liver mitochondria to yield carbamoylphosphate initiating the urea cycle,
Metabolic alkalosis the major mechanism of ammonium removal in humans. Healthy kidney produces
Glutamate dehydrogenase ammonium which may be released into the systemic circulation or excreted into the
Glutamine synthetase urine depending predominantly on acidbase status, so that metabolic acidosis increases
Glutaminase urinary ammonium excretion while metabolic alkalosis induces the opposite effect. Brain
Hyperammonemia and skeletal muscle neither remove nor produce ammonium in normal conditions, but they
are able to seize ammonium during hyperammonemia, releasing glutamine. Ammonia in
gas phase has been detected in exhaled breath and skin, denoting that these organs may
participate in nitrogen elimination. Ammonium homeostasis is profoundly altered in liver
failure resulting in hyperammonemia due to the deficient ammonium clearance by the
diseased liver and to the development of portal collateral circulation that diverts portal
blood with high ammonium content to the systemic blood stream. Although blood
ammonium concentration is usually elevated in liver disease, a substantial role of
ammonium causing hepatic encephalopathy has not been demonstrated in human
clinical studies. Hyperammonemia is also produced in urea cycle disorders and other
situations leading to either defective ammonium removal or overproduction of ammonium
that overcomes liver clearance capacity. Most diseases resulting in hyperammonemia and
cerebral edema are preceded by hyperventilation and respiratory alkalosis of unclear origin
that may be caused by the intracellular acidosis occurring in these conditions.
2012 Elsevier Inc. All rights reserved.

Abbreviations: NH3, ammonia; NH+4, ammonium ions; AST, aspartate aminotransferase; ALT, alanine aminotransferase; BCAT,
branched-chain amino acids aminotransferase; KGA, kidney-type glutaminase; GAC, glutaminase C; GAM, glutaminase M; GDH1,
glutamate dehydrogenase-1; GDH2, glutamate dehydrogenase-2; NAGS, N-acetylglutamate synthase; CPS1, carbamoylphosphate
synthetase-1; OTC, ornithine transcarbamylase; ASL, argininosuccinate lyase; ORNT, mitochondrial ornithine transporter; HHH,
hyperornithinemia, hyperammonemia and homocitrullinuria; IMP, inosine monophosphate; ALF, acute liver failure; UCD, urea cycle
disorder; CPT1, carnitine palmitoyltransferase-1; CPT2, carnitine palmitoyltransferase-2; CACT, carnitine-acylcarnitine translocase;
MCAD, medium-chain acyl-CoA dehydrogenase.
Corresponding author. Hospital General Juan Cardona, c/ Pardo Bazn s/n, 15406 Ferrol, La Corua, Spain. Tel.: +34 664 527 257;
fax: +34 981 17 81 59.
E-mail address: madevaa@yahoo.com (M.M. Adeva).

0026-0495/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.metabol.2012.07.007
1496 M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 1 (2 0 1 2) 1 49 51 51 1
Molecular nitrogen (N2) present in the earth atmosphere has
to be reduced to ammonia (NH3) by nitrogen-fixing bacteria
living independently in the soil or in the root of leguminous
plants before it may be utilized by humans. Ammonia
dissolves in water to form ammonium ions (NH4+) and this
form of reduced nitrogen is assimilated into amino acids and
other nitrogen-containing molecules. In aqueous solutions,
ammonia is a base (any compound accepting hydrogen ions)
forming a conjugated pair with the ammonium ion, according
to the reversible reaction: NH3 + H+ NH4+.
The pKa of the reaction is 9.3, indicating that at this pH
value, the concentration of the ionized (NH4+) and unionized
(NH3) forms is equal. When the pH of the solution is less than
9.3, hydrogen ions are incorporated to ammonia to yield
ammonium ions. Therefore, at physiological plasma and
intracellular pH values, virtually only the protonated moiety
(NH4+) is present in aqueous solutions [1]. The concentration of
ammonium in normal human plasma ranges between 11 and
50 mol/L and varies slightly in venous, arterial or capillary
blood. Free ammonium ions are continually produced and
consumed during cell metabolism in human body tissues.
They arise during the breakdown of purine and pyrimidine
derivatives, polyamines, and deamination of several amino
acids, including glutamine, asparagine, serine, threonine,
glycine, histidine, lysine, proline, hydroxyproline, homocys-
teine, and cystathionine [2]. Some free ammonium ions may
occasionally be supplied by urease-producing urea-splitting
organisms present in saliva [3], gastrointestinal tract [4,5],
urine [6], or other locations [7]. Free ammonium ions are
principally consumed to produce glutamine in the cytosol of
some human cells (principally skeletal muscle cells, hepato-
cytes, keratinocytes, gastrointestinal cells, lymphocytes, and
astrocytes) [8] and to generate carbamoylphosphate predom-
inantly inside liver mitochondria [9]. Ammonia in gas phase
has been detected in human skin [10] and exhaled air [11].
The enzymes primarily involved in the metabolism of free
ammonium ions are the cytosolic enzyme glutamine synthe-
tase [8] and the mitochondrial enzymes glutaminase [12] and
glutamate dehydrogenase [13]. In a reaction similar to the
glutaminase reaction, asparaginase yields free ammonium
ions and aspartate from asparagine [2]. In addition, the
enzyme carbamoylphosphate synthetase-1 catalyzes the
condensation of bicarbonate and ammonia to form carba-
moylphosphate inside the mitochondrial matrix, starting the
urea cycle in the liver [9]. Aminotransferases (transaminases)
are both cytosolic and mitochondrial enzymes engaged in
transferring amino groups between amino acid and keto acid
pairs, without generating or consuming free ammonium ions.
Important human transaminases include aspartate amino-
transferase (AST), alanine aminotransferase (ALT) and
branched-chain amino acids aminotransferases (BCAT) [2].
1. Glutamine synthetase
Glutamine synthetase is a cytosolic enzyme that catalyzes the
synthesis of glutamine from glutamate and free NH4+ in an
ATP-dependent reaction (Fig. 1). The human gene encoding
glutamine synthetase has been mapped to chromosome 1q25.
Related genes of unclear significance lie on chromosomes 5, 9,
and 11 [8,14]. Glutamine synthetase expression and activity
have been detected in adult human skin, peripheral lympho-
cytes, liver, brain (predominantly in astrocytes), and the
gastrointestinal tract, where the activity is highest in the
stomach, although the esophagus and both small and large
intestine have little synthesizing capacity [15,16]. In human
skin, glutamine synthetase expression is predominantly
associated with developing keratinocytes. It is present in all
layers of epidermis in young persons and primarily in the
stratum granulosum of elderly persons [17]. Dexamethasone
and the exposure to ammonium ions strongly induce the
activity of glutamine synthetase in spontaneously immortal-
ized human keratinocytes [17]. Glutamine synthetase expres-
sion is also remarkably induced by glucocorticoids in human
osteoblastic-like cells, while vitamin D inhibits basal and
glucocorticoid-stimulated glutamine synthetase activity by
affecting both the mRNA and protein levels of the enzyme [18].
In astrocytes, the activity of the enzyme is suppressed by
increasing the ADP concentration, which may be expected to
occur when the energy level of the cell is reduced [16].
Glutamine synthetase is present at early gestational stages
in human fetuses and placenta [19,20]. Human skeletal
muscle is capable of synthesizing glutamine, both during the
postprandial period [21,22] and the postabsorptive state [23].
Skeletal muscle of healthy individuals also synthesizes
glutamine following an intravenous infusion of leucine [21]
or a mixture of amino acids not containing glutamine [24].
Congenital deficiency of glutamine synthetase has been
rarely reported. A clinical picture with severe brain malforma-
tions, neonatal seizures, blistering skin lesions, multiorgan
failure, and frequent neonatal death has been associated with
homozygous mutations on the glutamine synthetase gene
[25,26]. Hyperammonemia has not been a consistent finding
in the few patients reported with congenital glutamine
deficiency [25,26]. The activity of glutamine synthetase was
markedly diminished with a concomitant reduction in the
amount of glutamine synthetase protein in the liver of two
patients who had fatal hyperammonemia after orthotopic
lung transplantation [27]. There may be a connection between
glutamine synthetase expression and some human tumors
associated with activating mutations in the gene encoding -
catenin, CTNNB1. In children, activating mutations in CTNNB1
occur in 80% of hepatoblastoma and 31% of nephroblastoma
tumors. In hepatoblastoma with activated -catenin, expres-
sion of glutamine synthetase is detected in tumor areas with
epithelial, but not with mesenchymal differentiation. Gluta-
mine synthetase expression was not observed in CTNNB1-
mutated nephroblastoma [28].
2. Glutaminase
Glutaminase is a phosphate-activated mitochondrial matrix
enzyme that catalyzes the hydrolysis of the amide group of
glutamine to stoichiometric amounts of glutamate and free
NH4+ (Fig. 2). Two human genes with considerable degree of
sequence similarity, GLS1 and GLS2, encode several glutamin-
ase isoenzymes. Human glutaminase-1 gene (GLS1) is located
on chromosome 2q32 and it is thought to encode an initial
mRNA transcript that undergoes tissue-specific alternative
 M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 1 (2 0 1 2) 1 49 5 1 5 11
Fig. 1 Glutamine synthetase reaction.
Fig. 2 Glutaminase reaction.
splicing generating three splice variants that are subsequently
translated into three human glutaminase isoenzymes: gluta-
minase-1 or kidney-type glutaminase (KGA), glutaminase C
(GAC), and glutaminase M (GAM). The human GLS1 gene spans
82 kb and is composed of 19 exons [12,29]. The full-length
crystal structure of human GAC has been recently determined
[30]. Human glutaminase-2 gene (GLS2) is located on chromo-
some 12q13 and encodes glutaminase-2 or liver-type gluta-
minase, a protein functionally similar to kidney-type
glutaminase but a little smaller in size [31]. Glutaminase
expression and activity have been detected in numerous
human tissues, including kidney, liver, brain (predominantly
in neurons), pancreas, airway epithelium, cardiac muscle,
skeletal muscle, small intestine, platelets, and fibroblasts.
Glutaminase-1 or kidney-type glutaminase is expressed
predominantly in kidney and brain [12,29]. Human airway
epithelial cells also express mRNA for KGA and they produce
ammonium ions stoichiometrically from glutamine [11].
Glutaminase C is expressed principally in pancreas and
cardiac muscle, but also appreciably in kidney, lung, and
placenta [12,29]. Human airway epithelial cells express mRNA
for GAC as well [11]. Glutaminase M is expressed in cardiac
and skeletal muscle [12]. Human tissue distribution of
glutaminase-2 or liver-type glutaminase is not well defined.
While normal human hepatocytes express liver-type gluta-
minase, hepatoma cells express the kidney-type isoform [29].
In the human gastrointestinal tract, glutaminase specific
activity is highest in the small intestine, intermediate in the
large intestine and lowest in the esophagus and stomach.
Therefore, both the small and large intestines have a high
potential for glutamine hydrolysis, unlike the stomach and
the esophagus [15]. Glucocorticoids enhance glutaminase
1497
Fig. 3 Asparaginase reaction.
Fig. 4 Glutamate dehydrogenase reaction.
activity, while interleukin-1, interleukin-6, tumor necrosis
factor (TNF)-, and interferon- have been shown to decrease
the activity of the enzyme in human cells [32]. Glutaminase
activity in human airway epithelial cells is enhanced in
response to acidic challenge while it is inhibited by interferon-
and TNF- [11]. Glutaminase may have a role in human cancer.
Human p53 gene exerts its tumor suppression action through
the transcriptional regulation of its target genes. Human
glutaminase-2 gene has been identified as a p53 target so that
p53 activation induces expression of GLS2 mRNA and therefore
GLS2 may contribute to the role of p53 in tumor suppression by
mediating some of its effects. GLS2 expression is lost or greatly
reduced in hepatocellular carcinomas and overexpression of
GLS2 reduces tumor cell colony formation [33,34]. On the other
hand, the oncogene Myc up-regulates glutaminase C expression
and promotes tumor cell proliferation in human B lymphoma
and prostate cancer cells [35] It appears that GLS1 and GLS2 may
have different roles in tumorigenesis. The tumor suppressor
gene p53 induces the expression of GLS2 but not GLS1 in various
cells, whereas the oncogene Myc induces the expression of GLS1
but not GLS2 [33].
3. Asparaginase
Analogous to the actions of glutaminase on glutamine,
asparaginase catalyzes the conversion of asparagine into
aspartate and a free ammonium ion (Fig. 3). Bacterial
asparaginase has been used for decades to treat some
lymphoproliferative disorders. It breaks down plasma aspar-
agine, interrupting the supply of this amino acid to cancer
cells present in the blood, which lack asparagine synthetase
and rely on blood-borne asparagine to survive. Asparaginase
administration may be associated with severe side-effects,
including hyperglycemia, hypertriglyceridemia due to in-
crease of endogenous synthesis of very low density lipopro-
teins, pancreatitis, hepatotoxicity (diffuse liver steatosis and
very rarely acute fulminant hepatic failure), acquired
1498 M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 1 (2 0 1 2) 1 49 51 51 1
antithrombin III deficiency, and thrombotic events [36].
Hyperammonemia has rarely been reported following aspar-
aginase use [37].
3.1. Glutamate dehydrogenase
Glutamate dehydrogenase is an enzyme that localizes pre-
dominantly to the mitochondrial matrix (and to a lesser
extent to the rough endoplasmic reticulum) and catalyzes the
reversible oxidative deamination of glutamate to produce -
ketoglutarate and a free ammonium ion [13]. The oxidative
deamination of glutamate uses NAD+ as cofactor and releases
H+ and the reduced compound NADH, whereas NADPH and
H+ are consumed in the synthetic reaction, releasing NADP+
(Fig. 4). Although reversible in the test tube, it is thought that in
vivo the glutamate dehydrogenase reaction proceeds predom-
inantly toward the direction of the oxidative deamination of
glutamate, producing -ketoglutarate and free ammonium
ions, probably due to the high concentration of glutamate and
the low level of free ammonium ions usually present inside the
mitochondria under baseline conditions. However, high con-
centration of ammonium ions and -ketoglutarate inside the
mitochondria might trigger biosynthesis of glutamate by
glutamate dehydrogenase [38,39].
Two highly homologous human genes, GLUD1 and GLUD2,
encode two glutamate dehydrogenase isoenzymes. GLUD1 is
located to chromosome 10q and encodes the isoenzyme
glutamate dehydrogenase-1 (GDH1). GLUD1 is a housekeeping
gene widely expressed in human tissues, including liver,
kidney, pancreatic -cells, brain, heart, intestine, spleen, skin,
lymph nodes, leukocytes, fibroblasts, and placenta. GLUD2 is
an intronless gene mapped to Xq chromosome that encodes
the glutamate dehydrogenase-2 isoenzyme (GDH2), being
predominantly expressed in retina, brain, and testicular tissue
in humans [38]. In testicular tissue, GDH2 is highly expressed
in Sertoli cells and to some extent in Leydig cells, while
spermatogonia and differentiated germ cells are negative for
this protein. In cerebral cortex, the expression of GDH2 is
restricted to astrocytes, with neurons showing only faint
immunoreactivity. Human liver does not express endogenous
GDH2 [40].
Glutamate dehydrogenase activity is regulated by allosteric
effectors. ADP and L-leucine are allosteric activators whereas
GTP, ATP, long-chain fatty acids (palmitoyl-CoA), estrogens,
epigallocatechin gallate (a polyphenol in green tea), and
NADH act as allosteric inhibitors [38,39]. The two glutamate
dehydrogenase isoenzymes differ in their allosteric regula-
tion. GDH1 activity is strongly inhibited by GTP while GDH2 is
resistant to the inhibitory effect of GTP, so that this isoenzyme
is able to metabolize glutamate even when the housekeeping
GDH1 protein is inhibited by sufficient amounts of GPT. In
addition, GDH2 depends on ADP for catalytic function. GDH2
maintains a very low baseline activity (less than 10% of its
capacity), its activation being dependent on rising ADP or L-
leucine levels [38,39]. Estrogens inhibit to a greater extent the
GDH2 isoenzyme than the GDH1 isoform. The inhibition of the
two isoproteins by estrogens is inversely related to their state
of activation induced by ADP [41]. Serum glutamate dehydro-
genase activity in healthy individuals is slightly gender-
dependent. The upper limit is 6.4 U/L for women and 11.0 U/
L for men [42]. Alcoholics have higher serum glutamate
dehydrogenase activity than normal individuals and the
enzyme activity decreases rapidly after cessation of drinking
[43,44]. Increased serum glutamate dehydrogenase activity in
alcoholics may be used as an index of the elapsed time from
the last alcohol intake [43,44]. Deficiency of glutamate
dehydrogenase activity has been associated with a poorly
defined neurological degenerative disorder with extrapyrami-
dal features (atypical Parkinson's disease), cerebellar dysfunc-
tion, peripheral neuropathy, and anterior horn cell signs,
suggesting that the phenotypic expression of the enzymatic
deficiency may be heterogeneous [45]. Gain of function
(activating) mutations on the GLUD1 gene increase GDH1
activity by rendering this isoenzyme resistant to the allosteric
inhibition by GTP while retaining the stimulatory action of
ADP and L-leucine, resulting in a constitutive enzyme
overactivity that leads to the hyperinsulinism hyperammo-
nemia syndrome [38,39]. Affected patients manifest hyper-
ammonemia and recurrent fasting or postprandial
hypoglycemia episodes related to enhanced insulin release
by pancreatic -cells thought to be due to an excess of
intracellular ATP. In the liver, it is believed that overactivity of
GDH1 leads to hyperammonemia by increasing free ammoni-
um ions generation due to unrestrained oxidation of gluta-
mate. Additionally, GDH1 overactivity might result in
glutamate depletion, reducing N-acetylglutamate formation
and consequently preventing urea cycle initiation [39].
3.2. Aminotransferases (transaminases)
Aminotransferases catalyze the reversible transfer of -amino
groups from -amino acids to -ketoacids without producing
or consuming free ammonium ions. The reversible transport of
amino groups between alanine/pyruvate, aspartate/oxaloacetate,
and branched-chain amino acids/branched-chain keto acids,
takes place via coupled reactions with the partner pair gluta-
mate/-ketoglutarate (Fig. 5). Alanine aminotransferase (ALT)
transports the -amino group from alanine to -ketoglutarate
with the generation of pyruvate and glutamate. Aspartate
aminotransferase (AST) catalyzes the transfer of -amino groups
from aspartate to -ketoglutarate with the formation of oxaloac-
etate and glutamate. The transfer of -amino groups from the
branched-chain amino acids (leucine, isoleucine, and valine) to
-ketoglutarate, generating their cognate branched-chain keto
acids (-ketoisocaproate, -keto--methylglutarate, and
-ketoisovalerate, respectively) and glutamate, is performed by
branched-chain amino acids aminotransferase (BCAT). Pyridox-
al phosphate, the functional form of vitamin B6, is a cofactor for
aminotransferases reactions.
3.3. Urea cycle enzymes
Human hepatocytes utilize two end-products of metabolism,
ammonia and carbon dioxide (under the form of bicarbonate),
to generate some amino acids (citrulline, arginine, and
ornithine) through a chain of reactions referred to as ornithine
cycle, KrebsHenseleit cycle or urea cycle (Fig. 6). Urea and
fumarate are produced while aspartate is consumed as a
result of the cycle functioning. Activity of the mitochondrial
isoenzyme of carbonic anhydrase is thought to be required to
 M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 1 (2 0 1 2) 1 49 5 1 5 11
Fig. 5 Aminotransferases reaction. ALT: alanine aminotransferase; AST: aspartate aminotransferase; BCAA: branched-chain
amino acids; BCAT: branched-chain amino acids aminotransferase; BCKA: branched-chain keto acids.
Fig. 6 Urea cycle. NAGS: N-acetylglutamate synthase; CPS1:
carbamoylphosphate synthetase-1; ORNT: mitochondrial
ornithine transporter; OTC: ornithine transcarbamylase;
ASL: argininosuccinate lyase; ASS1: argininosuccinate
synthetase-1. NAGS, CPS1, and OTC reactions take place in
the mitochondrial matrix. ASS1, ASL, and arginase-1
reactions occur in the cytosol.
supply bicarbonate from carbon dioxide, as urea synthesis in
healthy humans is inhibited by acetazolamide [46]. Amino
groups derived from various amino acids are funneled by
hepatic aminotransferases to glutamate, which is deaminated
in the mitochondria by glutamate dehydrogenase, producing
free ammonium ions and -ketoglutarate. Some amino acids
and other nitrogen-containing compounds do not participate
in transamination reactions, being directly deaminated to
generate free ammonium ions. When the rate of amino acid
catabolism increases, N-acetylglutamate is also synthesized
1499
from glutamate and acetyl-CoA by N-acetylglutamate
synthase, activating the enzyme carbamoylphosphate syn-
thetase-1 to initiate the urea cycle. Briefly, bicarbonate and
ammonia are condensed in the mitochondrial matrix of
hepatocytes to produce carbamoylphosphate by carbamoyl-
phosphate synthetase-1. Carbamoylphosphate is combined
with ornithine entering from the cytoplasm by ornithine
transcarbamylase to produce citrulline, which exits the
mitochondria. In the cytosol, the condensation of citrulline
and aspartate by argininosuccinate synthetase-1 yields argi-
ninosuccinate, which is transformed into arginine by argini-
nosuccinate lyase, liberating the carbon skeleton of aspartate
in the form of fumarate. Arginine is split by arginase-1
producing ornithine and urea. Ornithine travels from the
cytosol to the mitochondrial matrix via the ornithine trans-
porter to complete the cycle. Several unarchived polymor-
phisms have been identified that may be implicated in the
inter-individual variation observed in urea cycle function [9].
3.3.1. N-acetylglutamate synthase
The formation of N-acetylglutamate from glutamate and
acetyl-CoA inside the mitochondrial matrix is catalyzed by
the enzyme N-acetylglutamate synthase (NAGS). N-acetylglu-
tamate is an obligatory allosteric activator of carbamoylpho-
sphate synthetase-1, being therefore required to initiate the
urea cycle. The human NAGS gene is located on chromosome
17q21.31, being expressed in adult liver and small intestine as
well as in fetal liver. The intestinal transcript is smaller in
size than the liver transcript. The NAGS gene is not expressed
in adult brain, colon, heart, kidney, lung, muscle, placenta,
spleen, stomach or testis [47,48]. The activity of human NAGS
is augmented by the presence of arginine [49] and inhibited
by xanthine and uric acid [50]. Deficiency of NAGS activity
may be congenital or secondary to poorly defined causes,
such as primary carnitine deficiency [47,51]. Inherited NAGS
deficiency is an autosomal recessive disorder whose
phenotypic expression is similar to carbamoylphosphate
synthetase-1 deficiency. Neonatal presentation with respi-
ratory alkalosis, hyperammonemia and coma usually occurs
when the disease-producing mutations abolish NAGS
enzymatic activity whereas patients with partial NAGS
deficiency may present later in life with less severe
manifestations including recurrent vomiting and neurobe-
havioral changes [52]. N-carbamylglutamate is a functional
analog of N-acetylglutamate that activates carbamoylpho-
sphate synthetase-1 and restores urea cycle function in
patients with NAGS deficiency [52]. The NAGS inhibition by
1500 M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 1 (2 0 1 2) 1 49 51 51 1
xanthine and uric acid is reversed by supplementation with
N-carbamylglutamate [50].
3.3.2. Carbamoylphosphate synthetase-1
The enzyme carbamoylphosphate synthetase-1 (CPS1) cata-
lyzes the condensation of bicarbonate and ammonia to form
carbamoylphosphate in the mitochondrial matrix, being depen-
dent on N-acetylglutamate for activity. The carbamoylphos-
phate synthetase reaction takes place at the expense of two
molecules of ATP and is essentially irreversible. The human
gene encoding CPS1 has been mapped to chromosome 2 [53]
and a compilation of 222 molecular changes has been reported
[54]. A gene coding a novel isoform of CPS1 that shows high
expression in human testicular tissue has been cloned [55].
CPS1 has been shown to be among the most antigenically
dominant proteins in human liver mitochondria [56]. Immuno-
histochemical and Western blot analyses reveal strong and
diffuse CPS1 expression within normal human small intestine
mucosa and small-intestinal adenomas while protein expres-
sion is lost in adenocarcinomas [57]. Congenital CPS1 deficiency
is an autosomal recessive disorder that usually presents during
the neonatal period with severe hyperammonemia and coma,
but a delayed onset form has also been observed [53]. Secondary
low CPS1 activity in liver has been reported in a patient with
hyperinsulinism hyperammonemia syndrome [58].
3.3.3. Ornithine transcarbamylase
Ornithine transcarbamylase (OTC) catalyzes the synthesis of
citrulline from carbamoylphosphate and ornithine that enters
the mitochondria from the cytosol. The human gene encoding
OTC is located on Xp21.1 [59,60]. OTC deficiency is an X-linked
disease with phenotypic heterogeneity that usually presents
with neonatal hyperammonemia, respiratory alkalosis and
cerebral edema, although it also may be diagnosed in
adulthood or discovered in asymptomatic patients with only
biochemical abnormalities. Episodes are usually triggered by
catabolism, such as infections, and pregnancy is an additional
risk [59,60].
3.3.4. Argininosuccinate synthetase-1
The enzyme argininosuccinate synthetase-1 (ASS1) combines
citrulline and aspartate in the cytosol of hepatocytes to
generate argininosuccinate. Citrulline and aspartate leave
the mitochondrial matrix and enter the cytoplasm through
the ornithine transporter and the aspartateglutamate carrier
(citrin), respectively. The gene encoding ASS1 is located on
chromosome 9q34.1 and mutations producing ASS1 deficien-
cy have been recently compiled [61]. Congenital deficiency of
ASS1 activity causes type 1 citrullinemia, an autosomal
recessive disorder with phenotypic variability that ranges
from severely affected patients with neonatal hyperammo-
nemia to asymptomatic adult individuals with biochemical
manifestations of the disease [61,62].
3.3.5. Argininosuccinate lyase
Argininosuccinate lyase (ASL) is a cytosolic enzyme that
catalyzes the breakdown of argininosuccinate to arginine and
fumarate. The human gene that encodes argininosuccinate
lyase is positioned on chromosome 7 [63]. ASL deficiency
results in argininosuccinic aciduria, an autosomal recessive
disease characterized by tissue accumulation of argininosuc-
cinate and excessive excretion of this compound in urine.
Similarly to other urea cycle disorders except arginase-1
deficiency, ASL deficiency leads to reduced arginine synthesis.
Patients with ASL deficiency share the acute clinical phenotype
of hyperammonemia, encephalopathy, and respiratory alka-
losis common to other urea cycle defects, but they also display
chronic neurological symptoms that seem to be inevitable in
spite of careful treatment of hyperammonemia, believed to be
caused by a combination of tissue specific deficiency of
arginine and/or elevation of argininosuccinate [64,65].
3.3.6. Arginase-1
Arginase is the enzyme that catalyzes the hydrolysis of arginine
to ornithine and urea. In humans, two isoenzymes of arginase
have been demonstrated. Arginase-1 is a cytosolic protein,
while arginase-2 is located to the mitochondrial matrix [66].
Arginase-1 protein has been found in human liver, erythrocytes,
granulocytes, kidney, brain, and gastrointestinal tract, whereas
arginase-2 is detected in kidney, brain, gastrointestinal tract,
and fibroblasts. Therefore, arginase-1 is the only isoenzyme
found in liver and red blood cells while kidney, brain, and
gastrointestinal tract express the two arginase isoproteins
[6769]. The human gene encoding arginase-1 has been mapped
to chromosome 6q23 [70]. Mutations on this gene cause
deficiency of arginase-1 activity and congenital argininemia, a
rare autosomal recessive disorder that shows phenotypic
variability, similarly to other urea cycle disorders. Most patients
experience progressive mental impairment and neurologic
manifestations such as spastic tetraplegia during childhood.
Neonatal hyperammonemia is uncommon but metabolic crisis
may occur later in life [7173].
3.3.7. Mitochondrial ornithine carrier (ORNT)
In humans, the transport of ornithine from the cytosol to the
mitochondrial matrix across the mitochondrial membrane is
carried out by the mitochondrial ornithine transporter
(ORNT). Mutations in the human mitochondrial ornithine
carrier-1 abolish its transport activity and cause the hyper-
ornithinemia, hyperammonemia and homocitrullinuria
(HHH) syndrome [74,75].
3.3.8. Citrin (aspartateglutamate carrier)
The aspartate export from the mitochondrial matrix to the
cytosol in the liver is performed by citrin, an aspartate
glutamate carrier that catalyzes a 1:1 exchange of aspartate
for glutamate and a proton, being therefore electrogenic. The
human gene encoding citrin (SLC25A13) has been localized to
chromosome 7q21.3. Mutations on the SLC25A13 gene produce
defective citrin activity reducing aspartate export from the
mitochondria to the cytosol and leading to type-2 citrulline-
mia, an adult-onset autosomal recessive disorder in which
aspartate is not available to generate argininosuccinate in the
liver [62]. Most patients with type 2 citrullinemia may exhibit
hepatic steatosis which is not accompanied by obesity or the
metabolic syndrome [76]. Type 2 citrullinemia has been
associated with high incidence of hepatocellular carcinoma
[77,78]. Citrin protein is reduced in lymphocytes isolated from
peripheral blood in patients with citrin deficiency, suggesting
an alternative diagnostic method for this disorder [79].
 M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 1 (2 0 1 2) 1 49 5 1 5 11
Recently, the analysis of the urine metabolome based on gas
chromatography/mass spectrometry has been reported to be a
reliable diagnostic tool for citrin deficiency, readily differenti-
ating this disease from other causes of hyperammonemia [80].
4. Ammonium metabolism in healthy humans
Liver, portal-drained viscera (including stomach, small and
large intestines, spleen, and pancreas), kidney, skeletal
muscle, brain, lung, skin, and red blood cells are involved in
ammonium homeostasis in the human body.
4.1. Liver
The role of the liver in maintaining normal ammonium
metabolism is crucial. In healthy humans, the ammonium
concentration measured in the portal vein has been found
higher than the ammonium concentration in the hepatic vein,
indicating that ammonium is used by the liver, which
substantially reduces the high ammonium content present
in portal blood [81]. The source of the elevated ammonium
concentration in the portal vein has not been entirely
elucidated. Ammonium release associated with glutamine
extraction has been observed by the small and large intestines
in humans, being slightly more pronounced in the jejunum
than in ileum and colon [81,82]. Pancreas metabolism might
contribute ammonium to the portal vein via the glutamate
dehydrogenase reaction while spleen contribution to portal
ammonium level has not been explored. Urea-splitting
bacteria living in the mouth and the gastrointestinal tract
(including Helicobacter pylori) may generate ammonium from
urea. Additionally, ammonium in a concentration ranging
from 45 to 240 mol/L (average 110 mol/L) has been detected
in the hepatic bile in healthy humans undergoing a cholecys-
tectomy for gallstones [83]. The fate of the ammonium present
in the bile is unclear, but it may be released into the intestinal
lumen being subsequently transported by the portal vein to
the liver. Human hepatocytes use ammonia and bicarbonate
to form carbamoylphosphate in the mitochondria, initiating
the urea cycle reactions. In individuals with normal liver
function, hepatocytes seem to have a remarkable functional
reserve, and urea synthesis per gram of liver tissue increases
rapidly following major hepatectomy, so that liver resections
of up to 70%80% are generally well tolerated and arterial
ammonium concentration remains unchanged. In contrast,
much smaller liver resections precipitate liver failure in
patients with underlying liver disease [84]. Normal human
liver also shows glutamine synthetase and glutaminase
activity, being therefore capable of glutamine synthesis and
hydrolysis [12,15,16,29,46].
4.2. Kidney
Human kidney also plays a fundamental role in ammonium
homeostasis. Normal kidney cells produce free ammonium
ions that are either excreted into the urine or released into the
renal vein. In healthy individuals under normal acidbase
balance conditions, total kidney ammonium production is
approximately half-divided between urine and venous blood
1501
and the quantity of ammonium released into the systemic
circulation approximates the amount of ammonium excreted
in the urine. The release of ammonium into the renal vein
represents a major source of the normal ammonium concen-
tration in blood [81,8589]. The total amount of ammonium
produced by the kidney and its partition into the renal vein or
the urine may be modified in response to acidbase balance
and potassium status and kidney function. In healthy in-
dividuals, ammonium chloride-induced acidosis is associated
with an increase in the total kidney ammonium production
and a significant rise in the urinary excretion of ammonium.
In contrast, metabolic alkalosis is associated with a marked
reduction in urinary ammonium excretion and a rise in the
ammonium released into the kidney venous blood [87,89,90].
In healthy humans, potassium depletion enhances kidney
ammoniagenesis and urinary ammonium excretion (likely
associated with a decrease in urinary potassium excretion).
Conversely, the administration of potassium supplements
decreases urinary ammonium excretion [87,89]. In patients
with chronic kidney failure, total renal ammonium produc-
tion is decreased in relation to the reduction of functioning
renal mass and consequently both urinary ammonium and
ammonium added to the renal vein are reduced. However,
the rate of ammonium production per unit of glomerular
filtration rate is fourfold greater in patients with chronic
kidney failure than in healthy controls [88]. Glutamine is a
major contributor to kidney ammonium production both
under normal acidbase conditions and metabolic acidosis,
but other amino acids such as glutamate, glycine, and proline,
may also contribute [87,88].
4.3. Skeletal muscle
In healthy individuals under basal conditions, there is no
significant net uptake or release of ammonium ions by resting
muscle [9194], but normal skeletal muscle releases ammoni-
um during exercise and healthy individuals show an increase of
ammonium concentration in the vein draining the exercising
muscles. In some studies, simultaneous elevation of ammoni-
um in either arterial blood or venous blood draining the
contralateral resting limb has not been observed [95,96]. The
precise origin and fate of the ammonium released by the active
muscles are unclear [1]. During heavy exercise, ATP is rapidly
consumed, rendering ADP. Two molecules of ADP are combined
in the adenylate kinase or myokinase reaction, re-synthesizing
ATP with AMP as a by-product. The AMP is broken down to
inosine monophosphate (IMP) and an ammonium ion by the
enzyme AMP deaminase or myoadenylate deaminase. Free
ammonium ions may then be released into the venous blood of
the exercising limb. It is believed that the IMP may be converted
into adenylosuccinate which in turn is transformed again into
AMP, completing the proposed purine nucleotide cycle func-
tioning in human skeletal muscle. Aspartate is consumed by
the cycle, while fumarate is produced [97,98]. During hyper-
ammonemia, skeletal muscle may become a major ammoni-
um-removing organ, particularly due to its large mass. The
intravenous administration of ammonium salts increases
peripheral uptake of ammonium in normal persons, although
considerable individual variation is observed and once reached
a threshold plasma ammonium level, additional rise in arterial
1502 M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 1 (2 0 1 2) 1 49 51 51 1
ammonium concentration may not be accompanied by a
significant increase in peripheral uptake [93].
4.4. Brain
In healthy humans, ammonium cerebral arterio-venous
difference is negligible, indicating that there is neither a
significant net uptake nor release of ammonium by the brain
[91,92]. However, the rate of cerebral ammonium utilization
increases as a linear function of the arterial ammonium level
and net uptake of ammonium ions by the brain is observed in
healthy humans during hyperammonemia [99,100].
4.5. Lung
Human airway epithelial cells display glutaminase activity
and produce ammonium ions from glutamine. Ammonia has
been detected in gas phase in the exhaled breath and alveolar
air of healthy humans [11].
4.6. Skin
The skin could be an important organ to dispose of ammoni-
um ions, as ammonia is present in gas emanated from the
skin surface of healthy persons and patients with liver
disease; its concentration being correlated with blood ammo-
nium concentration [10,17].
4.7. Red blood cells
Human Rh proteins expressed in erythroid cells and epithelial
tissues have been defined as ammonium transporters,
although their precise localization, carrier mechanisms, and
clinical significance are yet to be clarified [101].
5. Ammonium metabolism in liver disease
Ammonium homeostasis and the interorgan trafficking of
ammonium are altered in hepatic disease. Similarly to healthy
persons, the portal-drained viscera produce ammonium in
patients with cirrhosis and the ammonium production is
related to glutamine uptake [102,103]. Hepatocellular dysfunc-
tion results in impaired clearance of ammonium by the liver.
In addition, patients with liver disease may develop portal
collateral veins that divert portal blood with high ammonium
content to the systemic circulation. Both the incomplete
clearance of ammonium and the development of collateral
portal circulation contribute to hyperammonemia generally
present in liver failure. The role of the portal collateral
circulation is suggested by studies showing that patients
with portal vein collateral circulation or portacaval anasto-
moses exhibit an increase in peripheral blood ammonium
concentration exceeding the concomitant rise in ammonium
values in hepatic vein blood following ingestion of ammoni-
um chloride [104,105]. Diminished ammonium clearance by
the cirrhotic liver is suggested by the high ammonium
concentration present in the hepatic vein in patients with
liver disease [103]. Urea synthesis capacity is reduced in
cirrhotic patients, the maximal rate ranging from 10% to 90%
of normal [46,106]. Similarly to healthy humans, acetazol-
amide inhibits urea synthesis in cirrhotic liver slices by about
50%, suggesting that hepatic urea synthesis depends on the
activity of mitochondrial carbonic anhydrase [46]. Glutamine
synthesis is also diminished in cirrhotic livers, compared
with normal controls. Conversely, the flux through hepatic
glutaminase is increased in cirrhosis 4- to 6-fold [46].
Compared to healthy individuals in whom ingestion of
amino acids does not induce hyperammonemia, blood
ammonium concentration in patients with hepatic cirrhosis,
particularly those with a transjugular intrahepatic portosys-
temic shunt, is transiently elevated following oral adminis-
tration of some amino acids, such as glutamine, glycine,
serine, and threonine, indicating a deficient handling of
ammonium in liver disease [102,107].
Similarly to healthy individuals, the kidney releases
ammonium into the renal venous circulation in patients
with liver disease and this delivery represents a major source
of the ammonium concentration in blood [100,103,108]. The
kidney is also largely responsible for the hyperammonemia
induced by administration of acetazolamide, chlorothiazide
and mercurial diuretics to patients with liver disease. The
intravenous administration of acetazolamide or chlorothia-
zide results in a significant elevation in the ammonium
released into the renal vein and a reduction in the ammonium
excretion in the urine that correlates with a rise in urine pH
[108,109]. Administration of mercurial diuretics to cirrhotic
patients also produces an elevation in arterial ammonium
concentration associated with an increase in the amount of
ammonium released into the renal venous blood while
extremities, liver, and brain do not contribute ammonium to
the circulation under these conditions. [110]. In addition,
voluntary hyperventilation is associated with hyperammone-
mia of unclear origin in patients with liver disease [86,111]. It
has been recently shown that the urinary ammonium
excretion increases during the anhepatic phase of a liver
transplantation and after reperfusion of the organ [112].
Skeletal muscle may contribute to ammonium clearance in
patients with liver disease and hyperammonemia. Unlike
healthy individuals, peripheral arterio-venous ammonium
concentration is slightly positive in patients with liver disease,
indicating that the skeletal muscle takes up ammonium in
these patients [91,94,100,103,113]. Further, the uptake of
ammonium by skeletal muscle rises with increasing levels of
blood ammonium [100]. However, the capacity of skeletal
muscle to extract ammonium from the systemic circulation is
reduced in cirrhotic patients with gross muscle wasting [94]. In
patients with hepatic insufficiency, the peripheral arterio-
venous difference for glutamine is threefold greater than that
for alanine, in contrast to healthy humans, in whom the
release of alanine exceeds that of glutamine, suggesting that a
major fraction of ammonium taken up by muscle is released
as glutamine in patients with liver disease [94]. In cirrhotic
patients, muscular exercise produces greater ammonium
release by skeletal muscle than in normal individuals,
resulting in larger increase in the venous ammonium
concentration of blood draining the exercising forearm [95,96].
Unlike healthy individuals, patients with hepatic disease
exhibit a positive cerebral arterio-venous difference in am-
monium concentration, indicating ammonium uptake by the
 M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 1 (2 0 1 2) 1 49 5 1 5 11
brain that shows a correlation to the level of ammonium
entering the brain in these patients [91,92,114].
5.1. Ammonium and hepatic encephalopathy
Advanced liver disease is usually accompanied by an
elevation of blood ammonium levels, but the role of
ammonium causing hepatic encephalopathy is controversial
and conclusive evidence of a major causative role of
hyperammonemia on hepatic coma has not been provided
in human clinical studies. There is a marked variability in the
blood ammonium level in patients with liver disease with and
without encephalopathy and overlap of blood ammonium
values between groups of patients at all levels of conscious-
ness is present. Furthermore, the correlation between the
blood ammonium concentration and hepatic encephalopathy
has not been consistent [94,104,115121]. In a prospective
study aimed to evaluate the correlation between plasma
ammonium levels and chronic hepatic encephalopathy, a
moderate correlation between blood ammonium level and
the severity of encephalopathy was found, but there
remains substantial overlap in blood ammonium concen-
tration between cirrhotic patients with and without hepatic
encephalopathy [121].
A number of clinical studies have analyzed the role of
ammonium as risk factor for brain edema in acute liver failure
(ALF). Univariate analyses suggest that the mean plasma
ammonium concentration in patients with ALF is higher in
patients with cerebral edema or brain herniation than in those
without these complications, but considerable overlap is
observed between the groups of patients [122,123]. In a large
cohort of patients with ALF, multivariate Cox regression
analysis revealed that the adjusted hazard ratios for elevated
blood ammonium concentration and risk for intracranial
hypertension and severe encephalopathy were 1.010 and
1.008, respectively, suggesting that other factors are addition-
ally important in producing brain edema during fulminant
hepatic failure [124]. Patients with higher ammonium level at
admission develop more complications, including cerebral
edema, renal failure, and need for ventilation, suggesting
that they may suffer more severe disease [125]. The persis-
tence of hyperammonemia instead of the ammonium level on
admission may be associated with intracranial hypertension
[124,126].
6. Hyperammonemia
Elevation of blood ammonium concentration occurs in a
variety of situations, including hepatocellular dysfunction,
development of portal collateral circulation, urea cycle disor-
ders, lysinuric protein intolerance, carnitine deficiency, medi-
um-chain acyl-CoA dehydrogenase deficiency, valproate
administration, organic acidemias, Reye's syndrome, infec-
tions with urea-splitting organisms, chemotherapy for hema-
tologic malignancies, lung transplantation, Barth syndrome,
and other conditions such as pyruvate carboxylase deficiency,
pyruvate dehydrogenase complex deficiency, hyperinsulinism
hyperammonemia syndrome, distal renal tubular acidosis,
ureterosigmoidostomy procedures, use of glycine solution as
1503
irrigant agent during transurethral resection of the prostate,
amino acid total parenteral nutrition, and administration of
drugs such as asparaginase, 5-fluorouracil, carbamazepine,
and topiramate. Most of the conditions inducing hyperammo-
nemia funnel to an imbalance between the amount of
ammonium produced and the body capacity to metabolize or
remove it, primarily by the liver. Congenital or acquired
defective urea cycle function leading to deficient ammonium
metabolic removal is a major cause of hyperammonemia, but
increased ammonium generation may overcome the hepatic
clearance capacity and produce hyperammonemia as well.
Tolerance of healthy persons to elevated blood ammonium
seems to be remarkable, as they may undergo a rise of arterial
ammonium concentration up to 510.7 mol/L without devel-
oping neurological alterations or electroencephalographic
abnormalities [85,93]. Similarly, symptoms suggesting hyper-
ammonemia have not been observed in patients affected
with hyperinsulinism hyperammonemia syndrome, despite
blood ammonium concentration that usually is 3- to 5-fold the
normal level [39]. Further, it has been recently shown that the
performance of psychological tasks is not affected by induced
hyperammonemia (up to 225 mol/L) in a double blind cross-
over study with healthy volunteers [127].
As previously outlined, severe liver disease and develop-
ment of portosystemic shunts usually result in hyperammo-
nemia due to defective hepatic ammonium clearance or
diversion of the portal blood with high ammonium content
into the systemic circulation.
6.1. Urea cycle disorders
Congenital or acquired deficiency of any of the enzymes
involved in the urea cycle reactions (N-acetylglutamate
synthase, carbamoylphosphate synthetase-1, ornithine trans-
carbamylase, argininosuccinate synthetase-1, argininosucci-
nate lyase, arginase-1, mitochondrial ornithine transporter,
and citrin) may produce hyperammonemia. Congenital defi-
ciency of the mitochondrial isoenzyme of carbonic anhydrase
has not been reported as cause of hyperammonemia.
Congenital urea cycle disorders (UCDs) are inherited as
autosomal recessive traits, except ornithine transcarbamylase
deficiency, which is X-linked. The most frequent congenital
UCD in Japan, US, and Finland is ornithine transcarbamylase
deficiency while the least frequent is arginase-1 deficiency.
The overall frequency of congenital UCDs is estimated to vary
from 1 in 30,000 live births to 1 in 50,000 live births
[64,74,128,129]. Patients with UCDs characteristically exhibit
an encephalopathy commencing with hyperventilation and
early respiratory alkalosis followed by coma and hyperam-
monemia. Approximately half of the cases manifest during
the neonatal period and these patients have poorer outcome
than those who present later in life. Most long-term survivors
with neonatal-onset UCD have intellectual disabilities which
become more pronounced with increasing age [64,74,128130].
However, even patients with partial urea cycle enzyme
deficiencies who manifest with late-onset UCDs demonstrate
evidence of neurocognitive and behavioral impairment,
including autism, learning disorders, and hyperactive and
self-injurious behavior. Even asymptomatic ornithine trans-
carbamylase deficient heterozygotes have been reported to
1504 M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 1 (2 0 1 2) 1 49 51 51 1
have cognitive deficits and are at risk for learning disabilities
and attention deficit hyperactivity disorder. Pregnancy, infec-
tious illnesses or fasting with subsequent catabolism, or the
use of sodium valproate may unmask a latent case of CPSI or
OTC deficiency [130,131]. For unknown reasons, arginase-1
deficiency and ornithine transporter deficiency differ from
other UCDs as the major neurological presentation is pyra-
midal tract involvement and spasticity, with hyperammone-
mic coma being rare [131]. There is no direct correlation
between the disease-causing molecular change, the peak
ammonium level, and the phenotypic manifestation, so that
prediction of neurological outcome is not straightforward.
There is a significant negative linear correlation between
duration of neonatal hyperammonemic coma and intelligence
quotient (IQ) at 12 months, suggesting that prolonged
neonatal coma is associated with brain damage and impair-
ment of intellectual function. However, there is no significant
correlation between peak ammonium level and IQ score at 12
months and a multivariate analysis indicates that the peak
ammonium level did not add to the significance of the
association between duration of coma and IQ at 12 months
[128]. Survivors who are severely mentally retarded have
chronic neuropathological findings, including increased ven-
tricular size and areas of focal cortical necrosis that may
reflect hypoxia and increased intracranial pressure [128,131].
In patients with ornithine transcarbamylase deficiency,
carbamoylphosphate synthetase-1 deficiency, and arginino-
succinate lyase deficiency, the development of neurological
symptoms seems to be inevitable despite aggressive therapy
and avoidance of hyperammonemia and virtually all survi-
vors have developmental disabilities. In these patients,
therapy fails to prevent severe neurological sequelae during
a hyperammonemic crisis [64,130].
6.2. Lysinuric protein intolerance
Lysinuric protein intolerance is an autosomal recessive
disorder caused by mutations in the SLC7A7 gene that induce
defective cationic amino acids (lysine, arginine, ornithine)
transport at the basolateral membrane of epithelial cells in the
intestine and kidney, resulting in hyperaminoaciduria, espe-
cially lysinuria, and in low plasma levels of arginine, ornithine
and lysine [132,133]. The highest incidence of lysinuric protein
intolerance has been found in Finland with more than half of
the reported cases coming from this country [133,134]. There
is high variability in the clinical presentation even within
individual families. Typically, the disorder presents in infancy
with feeding difficulties, vomiting, diarrhea, and poor growth,
but it may also present in adult life. Postprandial hyperam-
monemia produces episodes of lethargy, convulsions, or
coma. Sometimes there is a variety of other symptoms,
including aversion to protein-rich foods, hepatosplenome-
galy, lens opacities, hyperextensible joints, hyperelastic skin,
osteoporosis, short stature, hemolytic anemia, pancytopenia,
neurological involvement, mental retardation, focal glomer-
ulosclerosis, renal tubular disease, interstitial lung disease
and pulmonary alveolar proteinosis [132,133,135]. Recurring
postprandial hyperammonemia is assumed to be due to a
depletion of ornithine and arginine in liver mitochondria.
Citrulline administration, which is metabolized sequentially
to arginine and then ornithine, results in improved urea cycle
performance and amelioration of the postprandial rise of
plasma ammonium [132,134].
6.3. Carnitine deficiency
Carnitine is a molecule normally derived from dietary
protein although it may be synthesized by the liver from
lysine and methionine as a methyl donor. It is required for
the transfer of medium- and long-chain fatty acids across
the mitochondrial membrane to be oxidized. Carnitine
enters the cell across a plasma membrane carnitine trans-
porter, OCTN2, being conjugated with fatty acids on the
outer mitochondrial membrane by carnitine palmitoyltrans-
ferase-1 (CPT1). The acylcarnitine complex is transferred
across the inner mitochondrial membrane by the enzyme
carnitine-acylcarnitine translocase (CACT). The fatty acid is
liberated inside the mitochondrial matrix by carnitine
palmitoyl transferase-2 (CPT2) for subsequent -oxidation,
while the carnitine molecule is returned to the outer
mitochondrial membrane by the translocase, ready for
another cycle of fatty acid transfer. Congenital deficiency
of OCTN2, CPT1, CACT, or CPT2 may result in hyperammo-
nemia of unclear origin [136139]. Deficient formation of
acetyl-CoA as a result of impaired fatty acid oxidation inside
the mitochondria may disrupt urea cycle function by
reducing N-acetylglutamate production. Primary carnitine
deficiency due to dysfunction of OCTN2 has been associated
with partial deficiency of the enzyme N-acetylglutamate
synthase [51]. Hyperammonemia associated with acquired
carnitine deficiency in a severely malnourished patient has
also been reported [140].
6.4. Medium-chain acyl-CoA dehydrogenase deficiency
The enzyme medium-chain acyl-CoA dehydrogenase
(MCAD) is involved in mitochondrial fatty acid oxidation,
being encoded by the human gene ACADM, located on
chromosome 1p31. Congenital MCAD deficiency is an
autosomal recessive disorder ranking as one of the most
frequent defects of fatty acid metabolism in the US [141].
Clinical features of MCAD deficiency may be triggered by
catabolic stress, such as infections and fasting, and include
sudden infant death, hypoketotic hypoglycemia and hyper-
ammonemia [142]. Disruption of the fatty acid oxidation
results in mitochondrial accumulation of unoxidized fatty
acyl-CoA metabolites that are believed to inhibit the urea
cycle [140,143].
6.5. Valproate therapy
Valproate administration has been consistently associated
with hyperammonemia, particularly when combined with
other anticonvulsant drugs, including phenytoin, phenobar-
bital, and topiramate. Serum levels of ammonium do not
correlate with the severity of valproate-induced encephalop-
athy and both asymptomatic hyperammonemia and carnitine
deficiency are common in patients receiving valproate.
Sometimes, valproate therapy has unmasked an underlining
urea cycle disorder [144146].
 M E TAB O LI S M CL I NI CA L A N D EX P ER IM EN T AL 6 1 (2 0 1 2) 1 49 5 1 5 11
6.6. Organic acidemias
Organic acidemias including propionic acidemia, methylma-
lonic acidemia, and isovaleric acidemia may result in
hyperammonemia. Amino acids such as methionine, threo-
nine, valine, and isoleucine, the side chain of cholesterol, and
odd chain fatty acids produce propionate in their degradation
pathways. Propionyl-CoA is sequentially transformed into
methylmalonyl-CoA and succinyl-CoA, which enters the
tricarboxylic acids cycle, serving as an anaplerotic com-
pound. Vitamin B12 is necessary for the conversion of
methylmalonate to succinate. Propionic acidemia and iso-
valeric acidemia are autosomal recessive disorders caused by
propionyl-CoA carboxylase deficiency and isovaleryl-CoA
dehydrogenase deficiency, respectively [147149]. Methylma-
lonic aciduria is a heterogeneous disorder that recognizes
both genotypic and phenotypic variability. It may be due to
defective cobalamine synthesis (responsive to vitamin B12) or
to mutations in the MMACHC gene located in chromosome
region 1p34-1, which produce combined methylmalonic
aciduria and homocystinuria or cobalamin C disease. In
a retrospective study of 30 patients with vitamin B12-
unresponsive methylmalonic aciduria, half of them have a
neonatal onset. Neurological deterioration and chronic kid-
ney failure are frequent manifestations of the disease [150].
Administration of N-carbamylglutamate, a carbamoylpho-
sphate synthetase-1 activator, improves hyperammonemia
associated with organic acidemias, suggesting that a dis-
rupted urea cycle may be the cause of the hyperammonemia
in these conditions [151154].
6.7. Reye's syndrome
Reye's syndrome occurs following an apparently uneventful
recovery from a viral illness and may be precipitated by
salicylate administration, being characterized by cerebral
edema, diffuse fatty infiltration of the viscera, and a liver
biopsy considered diagnostic of the syndrome. Reye's syn-
drome also features hyperammonemia of unclear cause
and increased blood concentration of fatty acids and lactic
acid [155].
6.8. Infections with urease-producing organisms
Urinary tract infections with organisms that produce urease
such as Proteus mirabilis, Klebsiella oxytoca, Klebsiella pneumo-
niae, and Corynebacterium urealyticum, may induce severe
hyperammonemia and coma, particularly in association
with urinary dilatation or dysfunctional neurogenic bladder.
Urease breaks down urea generating ammonium which is
released into the systemic circulation and may exceed liver
clearance capacity, resulting in hyperammonemia [6,156,157].
Pelvic abscesses have also been rarely associated with
hyperammonemia [7].
6.9. Chemotherapy for hematologic malignancy
Severe hyperammonemia preceded by hyperventilation and
respiratory alkalosis has been repeatedly observed in patients
receiving chemotherapy for hematologic malignancy and
1505
following bone marrow transplantation [158] or stem cell
autograft [159]. Necropsy findings observed in these patients
include ischemic changes in cerebral cortex and cerebral
edema with astrocyte swelling. In the liver, sinusoidal
dilatation, marked acute congestion, hemosiderin deposition,
and centrilobular fat infiltration are found. In the lungs,
hemorrhage and edema along with bilateral pleural effusion
are present. Other findings include pericardial effusion,
peritoneal effusion, hemorrhagic cystitis, and ischemic colitis
[159,160].
6.10. Lung transplantation
Hyperammonemia has been noted in 4% of patients following
lung transplantation and its presence has been associated
with increased mortality [161]. Risk factors for hyperammo-
nemia in lung transplant recipients include development of
major gastrointestinal complications, use of total parenteral
nutrition, and lung transplantation for primary pulmonary
hypertension [161].
6.11. Barth syndrome
Barth syndrome is a rare X-linked disorder due to mutations on
the TAZ gene, characterized by skeletal myopathy, cardiomy-
opathy, left ventricular noncompaction, neutropenia, growth
retardation, and 3-methylglutaconic aciduria. The TAZ gene is
located in Xq28 and its gene product, taffazin, is probably
involved in cardiolipin synthesis, as the concentration of this
phospholipid is markedly decreased in skeletal and cardiac
muscle and in platelets from affected patients. Barth syndrome
may be accompanied by acute metabolic decompensation,
lactic acidosis and hyperammonemia of unclear cause [162,163].
6.12. Other causes of hyperammonemia
Hyperammonemia has been reported to be associated with
pyruvate carboxylase deficiency (presumably due to shortage of
aspartate supply to the urea cycle) [164], pyruvate dehydrogenase
complex deficiency (attributed to defective acetyl-CoA synthesis
and secondary inhibition of ureagenesis) [165], hyperinsulinism
hyperammonemia syndrome [39], distal kidney tubular acidosis
(assumed to be due to defective urinary ammonium excretion)
[166], ureterosigmoidostomy [167], essential amino acid total
parenteral nutrition [168], and drugs including asparaginase [37],
5-fluorouracil [169], carbamazepine [170], topiramate [145], and
glycine solution used as an irrigant [171].
7. Hyperammonemia, cerebral edema, and
respiratory alkalosis
Hyperventilation and consequent respiratory alkalosis usually
precede or accompany the acute metabolic crisis with cerebral
edema and coma typical of most diseases featuring hyper-
ammonemia. Indeed, any alteration in the level of conscious-
ness associated with respiratory alkalosis should prompt the
determination of blood ammonium level [128,130]. Respiratory
alkalosis is the predominant acidbase anomaly in patients
with liver disease, particularly in those with cerebral edema
1506 M ET AB O LI S M CL I NI CA L A N D EX PE R IM EN T AL 6 1 (2 0 1 2) 1 49 51 51 1
and hepatic coma [120,172174] and it has been suggested that
cirrhotic patients with simultaneous hyperammonemia and
reduction in the plasma partial pressure of carbon dioxide
(pCO2) are more likely to be affected by cerebral edema and
coma [120,172]. Hyperventilation is also an early sign of the
acute metabolic decompensations associated with urea cycle
disorders and other diseases featuring hyperammonemia,
being followed by an encephalopathy characterized by cerebral
edema and coma. Tachypnea and respiratory alkalosis pro-
gressing to cerebral edema and coma with hyperammonemia
have been reported in ornithine transcarbamylase deficiency
[59], argininosuccinate synthetase deficiency, argininosucci-
nate lyase deficiency [65], lysinuric protein intolerance [132],
medium-chain acyl-CoA dehydrogenase deficiency [142], pro-
pionic acidemia [175], valproate administration [144], Reye's
syndrome [176], urinary tract infections with urea-splitting
organisms [6,156,157], chemotherapy for the treatment of
hematologic malignancy [160], bone marrow transplantation
[160], stem cell autograft for multiple myeloma [159], infusion of
essential amino acid total parenteral nutrition [168], and
5-fluorouracil administration [177].
The cause of hyperventilation in these disorders is un-
known. In humans, the central nervous system controls
respiration via unclear mechanisms. The respiratory control
center in the medulla oblongata likely receives input from
adjacent chemoreceptors which sense changes in blood
hydrogen ion concentration and pCO2. In patients with hepatic
cirrhosis, it has been suggested that ammonium ions might
induce ventilatory stimulation. Another possible mechanism
that may explain hyperventilation in this condition is the
intracellular acidosis that accompanies liver cirrhosis [174].
Hepatic failure is associated with elevated plasma concentra-
tion of lactate, pyruvate, and other organic anions, resulting in
intracellular acidosis [116,120,173,174,178,179]. Furthermore, a
rise in the erythrocyte concentration of 2,3-bisphosphoglyce-
rate [180182] and a displacement to the right of the
oxyhemoglobin dissociation curve [174,181,183] have been
observed in patients with liver disease, supporting the notion
that tissue acidosis induces a reduction in the affinity of
hemoglobin for oxygen aimed to improve carbon dioxide
extraction from tissue cells. Similarly to hepatic failure, most
disorders leading to hyperammonemia and acute metabolic
decompensations with cerebral edema are also characterized
by intracellular acidosis due to accumulation of different
organic anions. Hyperventilation may be an adaptative
physiological response to intracellular acidosis intended to
enhance carbon dioxide removal, as intracellular pH reflects
cellular metabolic status better than blood pH [184].
In this review, we have summarized relevant articles
concerning the enzymes involved in human ammonium
metabolism, the homeostasis of ammonium ions in healthy
humans and patients with liver disease, and the main causes
of hyperammonemia. We additionally discuss the occurrence
of hyperventilation and respiratory alkalosis associated with
cerebral edema and hyperammonemia. Although a thorough
search for relevant articles in the medical literature has been
performed, the broad scope of the topic makes it difficult to
expand the content of the information in every section. The
knowledge regarding the human distribution and activity of
the enzymes involved in ammonium metabolism and the
molecular basis of the different disorders causing hyperam-
monemia has fundamental translational potential in order to
establish the pathogenic mechanisms and the therapy for
these diseases.
Authors contributions
M. Adeva and G. Souto carried out the literature search and
wrote the draft of the manuscript. N. Blanco reviewed urea
cycle disorders. Ammonium metabolism in healthy humans
and liver disease was reviewed by C. Donapetry. M. Adeva and
G. Souto reviewed glutamine synthetase, glutaminase and
glutamate dehydrogenase and hyperammonemia. All authors
contributed to the final version of the manuscript.
Funding
There was no financial support for this work.
Conflict of interest
There are no conflicts of interest.
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