Download Clinical Application of Blood Ammonia Determinations

Clinical Application of Blood
Ammonia Determinations
C/E Update:
Chemistry III
by Allen M. Glasgow, M.D.
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LABORATORY MEDICINE.
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Introduction
This article briefly reviews ammonia toxicity and ammonia metabolism. Disorders of ammonia
metabolism are described. Methods
for measuring plasma ammonia
are then described, followed by a
discussion of the technical difficulties of ammonia assays. The
article concludes with a discussion
of alternate methods for assessing
ammonia metabolism.
Teleologic Problem
Ammonia is a waste product of
protein and amino acid metabolism.
In aquatic animals, ammonia is
easily disposed of by simple diffusion into the water. As life evolved
onto land, it became necessary to
From the Department of Endocrinology
and Metabolism, Children's Hospital National Medical Center, Washington, D. C.
develop other means of eliminating
ammonia. Birds excrete waste
nitrogen as uric acid, which can be
highly concentrated, thus enabling
them to conserve water and keep
their weight low. Excess nitrogen
in mammals is eliminated as urea.
Ammonia as a Toxin
A l t h o u g h elevated a m m o n i a
levels affect the metabolism of a
variety of tissues, the toxic effects
on the central nervous system
dominate the clinical picture in
hyperammonemia. The clearest
clinical example of this toxicity is
seen in the inherited disorders of
the urea cycle.
The mechanism of ammonia
neurotoxicity is not well understood. An elevated neuronal ammonia may shift the equilibrium of
the reaction sequence:
Alpha-ketoglutarate + NH 4 +
+ NADH -» Clutamate
+ NAD + + H 2 0
Glutamate + NH 4 + —> Glutamine
It has been postulated that this
shift may decrease the concentration of alpha-ketoglutarate and
thus impair the activity of the
cerebral citric acid cycle. 1 - 3 Alternately, the elevated glutamine
may be converted, in part, to alphaketoglutaramate, which is neurotoxic. 4 Another possibility is that
the neurotoxicity of ammonia may
result from an alteration of neuronal
ionic equilibrium or altered neurotransmitter function. 5
Hyperammonemia causes both
severe reversible cerebral dysfuction and chronic irreversible
cerebral impairment. Severe, even
p r o f o u n d , cerebral dysfunction
may be reversible if acute hyperammonemia can be treated successfully. O n the other hand,
chronic mild hyperammonemia
without immediate symptoms may
cause irreversible cerebral damage.7
Acute hyperammonemia leads to
lethargy, vomiting, agitation, seizures, coma and eventual loss of
brainstem functions. Hyperventilation leading to a respiratory
alkalosis is c o m m o n in hyperammonemic states, presumably due
to a stimulation of the central respiratory center. The major effect
of chronic hyperammonemia is
impairment of intellectual function.
Ammonia Metabolism
Production
When considering ammonia
p r o d u c t i o n , it is better to think of
the " a m m o n i a " or nitrogen load
rather than free ammonia product i o n . Some " a m m o n i a " is transported to the liver as alanine,
glutamine and other amino acids.
While this " a m m o n i a " may never
appear in the plasma as free
ammonia, it is in equilibrium with
free ammonia and constitutes part
of the total ammonia load.
Ultimately, ammonia comes from
two sources: 1) ingested nitrogen
(primarily protein); and 2) hydrolysis of urea in the gastrointestinal tract. The latter source
0007-5027/81/0300/151 $00.85 © American Society of Clinical Pathologists
151
contributes to the ammonia load
because the ammonia must be
reconverted to urea. Since 30% to
40% of the urea produced in the
liver is hydrolysed to ammonia in
the intestine, 8 the total ammonia
load is about 30% to 40% greater
than ingested nitrogen, minus
small amounts of nitrogen eliminated by mechanisms other than
the urea cycle. These mechanisms
include net protein synthesis in
growing children, skin loss, ammonia in expired air, stool nitrogen,
and nitrogen excreted as creatinine,
uric acid and urinary ammonia. In
general, the nitrogen eliminated
by these mechanisms is small
compared with the ammonia load.
In addition, the amount is relatively constant and not influenced
by nitrogen intake.
GLUTAMINE + C O ,
Carbamyl phosphate is also produced in the cytoplasm by another
carbamyl phosphate synthetase
152
GLUTAMATE
I
OROTIC A C I D
ORNITHINE
CITRULLINE
PYRIMIDINES
\9
CYTOPLASM
/So
MITOCHONDRIA
f
ORNITHINE
CITRULLINE
ASPARTATE
UREA
ARGININOSUCCINATE
The Urea Cycle
The complete urea cycle (Fig. 1)
is present only in the liver. The
first step of the urea cycle is
catalyzed by mitochondrial carbamyl phosphate synthetase. A m monia, C 0 2 and t w o ATPs are
converted to carbamyl phosphate,
phosphate and two ADPs. Mitochondrial carbamyl phosphate
synthetase is inactive in the absence of N-acetyl glutamate. Nacetyl glutamate is f o r m e d f r o m
acetyl CoA and glutamate by glutamate N-acetylase, which is activated
by arginine. The activity of this
enzyme is also increased in animals
that are fed a high-protein diet,
suggesting that increased protein
intake is one way in which the urea
cycle is regulated. 9 An inheritable
deficiency of glutamate N-acetylase
has never been described, but it is
likely that such a deficiency w o u l d
produce a disorder similar to the
urea cycle enzyme deficiencies
discussed below.
ACETYL COA
CAR BAM YL PHOSPHATE " ^ t .
ARGININE
FUMARATE
Fig. 7. The urea cycle and related reactions (see text). Key to enzymes and
transport processes: 7 = carbamyl phosphate
synthetase
(mitochondrial); 2 = ornithine
transcarbamylase;
3 = argininosuccinate
synthetase; 4 = argininosuccinate
lyase; 5 = argininase; 6 = glutamate
Nacetylase; 7 = ornithine
aminotransferase;
8 = carbamyl
phosphate
synthetase (cytoplasmic); 9 = ornithine transport process; 10 = citrulline
transport process.
which utilizes glutamine rather
than ammonia as a preferred substrate and does not require Nacetyl glutamate as an activator.
Cytoplasmic carbamyl phosphate
is part of the pyrimidine synthetic pathway. Normally, the
mitochondrial (urea cycle) pool of
carbamyl phosphate seems to be
separate from the cytoplasmic
(pyrimidine) pool. However, when
mitochondrial carbamyl phosphate
accumulates (as in ornithine transcarbamylase deficiency), carbamyl
phosphate diffuses into the cytoplasm, leading to an overproduction of orotic acid and other
pyrimidines.
LABORATORY MEDICINE • VOL. 12, NO. 3, MARCH 1981
The second urea cycle enzyme,
ornithine transcarbamylase, combines carbamyl phosphate and
o r n i t h i n e , forming citrulline and
releasing phosphate. This reaction
occurs in the mitochondrial matrix.
Ornithine enters the mitochondria
by a specific transport process. 10
There is also a specific transport
process for moving citrulline out
of the mitochondria. 1 0
The remainder of the urea cycle
enzymes, which regenerate ornithine f r o m citrulline and release
urea, are cytoplasmic. The first
cytoplasmic step catalysed by
argininosuccinate synthetase converts citrulline and asparatate t o
argininosuccinate, while consuming the energy of one high-energy
phosphate bond of ATP. Argininosuccinate lyase then catalyzes the
formation of arginine and fumarate
from argininosuccinate. Ornithine
is regenerated from arginine by
argininase, releasing urea. The net
effect of the sequence of reactions
is that ammonia, aspartate, C 0 2
and three ATPs are converted t o
urea, fumarate and three ADPs.
centration is normally about 300
/xmol/L, whereas the plasma ammonia concentration is normally
about 30 /umol/L. Thus, small rises
in alanine and giutamine can
" b u f f e r " relatively large rises in
ammonia. While such reactions
limit the rise in blood ammonia,
they do not result in the elimination of ammonia.
The proper functioning of the
urea cycle requires adequate levels
of ornithine. If dietary arginine is
low, ornithine must be produced.
If dietary arginine and ornithine
are in excess, they can be converted to glutamate. Production
and degradation of ornithine occurs
by a two-step process catalyzed by
ornithine aminotransferase and
glutamate semialdehyde dehydrogenase. These t w o enzymes,
rather than ornithine transcarbamylase, are probably the major
determinants of the hepatic ornithine level. In man, dietary arginine
appears to be in excess normally,
and the physiologic direction of
this sequence is toward glutamate.
Hyperornithinemia with gyrate
atrophy of the choroid and retina
is the only known disorder associated with hypoammonemia." The
hyperornithinemia is due to a
deficiency of ornithine aminotransaminase activity.
Amination Reactions
An acute change in ammonia
level may be temporarily "buffered"
by incorporating ammonia into
amino acids. This buffering may
be important in limiting fluctuations in tissue ammonia levels.
Incorporation of ammonia into
alanine and giutamine seems to be
most important physiologically.
Although it has been proposed
that the formation of giutamine
from alpha-ketoglutarate may contribute to ammonia toxicity, it is
equally likely that the formation of
giutamine and alanine is protective.
The serum alanine concentration is normally about 200 /umol/L
and the serum giutamine con-
Disorders of Ammonia Metabolism
Hypoammonemia
Hyperammonemia
Because of the body's large
reserve capacity for urea synthesis,
there are few examples of hyperammonemia due to increased production of ammonia. Infection of
an obstructed urinary tract with
Proteus, a urea-splitting organism,
has been reported to cause hyperammonemia. 1 2 The hyperammonemia in Reye's syndrome may be
due in part to increased ammonia
production. 1 3
Generalized Liver Disease
Hyperammonemia may occur in
any patient with severe liver disease including acute hepatitis,
cirrhosis or Reye's syndrome.
Hyperammonemia u n d o u b t e d l y
contributes to the encephalopathy
in these disorders; however, other
metabolic abnormalities due to
the hepatic dysfunction also play a
role. Hyperammonemia is relatively less important in acute fulminant hepatic failure than in
chronic hepatic encephalopathy
and Reye's syndrome. The e n cephalopathy of fulminant hepatic
failure will often respond tem-
porarily to L-dopa, suggesting that
the encephalopathy is partly due
to false neurotransmitters. 1 4
In patients with chronic liver
disease, the encephalopathy is
usually made worse by measures
that increase blood ammonia and
improved by measures that decrease blood ammonia. 16,17 O n the
other hand, the correlation between the severity of the encephalopathy and the blood ammonia level is not strong. Increased levels of plasma aromatic
amino acids and decreased levels
of plasma branched-chain amino
acids may also contribute to the
encephalopathy of chronic liver
disease.18 Increased aromatic amino
acids in the brain may increase the
formation of serotonin and other
neurotransmitters.
In Reye's syndrome, there is a
reasonably good correlation between the blood ammonia levels
at admission and the severity of
the encephalopathy. 1 9 However,
hepatic function invariably improves and the ammonia level
falls, even in patients w h o eventually die from this disorder. Patients
with Reye's syndrome have a
marked increase in ammonia production and probably also have
impaired hepatic ureagenesis. 13
Although the cause of the i m paired ureagenesis is not certain,
there is a transient decrease in
hepatic carbamyl phosphate synthetase and ornithine transcarbamylase activity.
Urea Cycle Disorders
Carbamyl phosphate
synthetase
(CPS) deficiency has been reported
in only a few patients. O n e patient
with complete CPS deficiency died
from overwhelming hyperammonemia shortly after birth. 2 0
Children with partial CPS deficiency may have severe mental
retardation, and may experience
episodic vomiting and lethargy
LABORATORY MEDICINE • VOL 12, NO. 3, MARCH 1981 1 5 3
following ingestion of high-protein
meals. 21
Ornithine transcarbamylase (OTC)
deficiency is the most c o m m o n
inheritable urea cycle disorder.
The gene for OTC is on the X
chromosome. 2 2 Males with OTC
deficiency have only the abnormal
gene, while females with OTC
deficiency have t w o genes, one
normal and one abnormal. The
most common mutation(s) of this
enzyme results in total loss of
activity. Affected males have no
OTC activity and usually die within
two to three days of birth. 2 3 Symptoms in females vary widely, depending on the balance between
the inactivation of the normal
and abnormal genes 24,25 : some
have severe mental retardation
and recurrent episodes of acute
encephalopathy; some have only
protein intolerance; still others
are asymptomatic. In rare cases,
male patients may have an OTC
mutation in which the enzyme
retains partial activity; these patients have symptoms similar t o
those of females.
Patients with OTC deficiency
excrete excessive amounts of
urinary orotic acid. The best way
to detect asymptomatic females
with OTC deficiency is to measure
the urinary orotic acid response t o
a protein load. 26
Citrullinemia
is caused by a
deficiency of argininosuccinate
synthetase. Complete deficiency
of this enzyme causes severe,
usually lethal, hyperammonemia
in infancy. Variants with partial
activity cause mental retardation
and episodic encephalopathy. 2 7
Plasma and urinary citrulline are
markedly elevated. It is not clear
if the hyperammonemia is due to a
"back u p " inhibition of OTC by
citrulline or a relative ornithine
deficiency.
Argininosuccinic aciduria causes
mental retardation and episodic
154
encephalopathy resembling the
milder forms of the disorders
described above. Some patients
with this disorder have short,
brittle hair due t o arginine deficiency. The hyperammonemia in
this disorder is caused by the
markedly increased excretion of
argininosuccinic acid, which leads
to an arginine and ornithine deficiency. 28
Argininemia is often associated
with modest elevations of blood
ammonia. Elevated arginine may
be more important than hyperammonemia in causing the athetosis and spasticity seen in patients with this disorder.
Disorders of Ornithine Metabolism
Lysinuric protein intolerance results from a defect in the transport
of dibasic amino acids in the
intestine, kidney and other tissues. 31,32 Poor a b s o r p t i o n , i n creased excretion and poor cellular
uptake of ornithine and arginine
lead to hyperammonemia because
of a deficiency of hepatic ornithine. This disorder, which is relatively common in Finland, usually
presents in infancy. The symptoms
of lysinuric protein intolerance are
failure to thrive, vomiting, diarrhea, hepatomegaly, osteoporosis
and hyperammonemia. Urinary
dibasic amino acids are increased,
and plasma levels tend to be low.
Arginine supplementation may
improve the hyperammonemia.
A disorder characterized by
ornithinemia,
hyperammonemia
has been
and homocitrullinuria
postulated to be due to a defect
in mitochondrial ornithine transport. 33 The few patients reported
with this disorder have been severely retarded. The hyperammonemia may respond to ornithine
supplementation. The h o m o c i trulline is presumably formed by
the replacement of ornithine with
lysine in the ornithine transcarbamylase step.
LABORATORY MEDICINE • VOL 12, NO. 3, MARCH 1981
may cause
Hyperalimentation
hyperammonemia if the administered amino acids contain relatively low levels of arginine.
Other Causes of Hyperammonemia
Most premature infants have
slightly higher blood ammonia
levels than full-term infants, although it is not known if this
increased level is deleterious. A
few preterm infants have marked
hyperammonemia that lasts t w o to
three days and is associated w i t h
severe neurologic symptoms. 3 3
The cause of this condition is
u n k n o w n . If the infant can be
supported, the prognosis is g o o d .
After recovery, ammonia metabolism appears to be normal.
Patients with a number of organic acidemias including propionic
acidemia, methylmalonic acidemia,
isovaleric acidemia and glutaric
acidemia may have hyperammonemia, especially during acute
episodes of acidosis. The hyperammonemia may be due to inhibition of the urea cycle by the
accumulating organic acid or the
coenzyme A derivative. Valproic
acid therapy may also cause mild
hyperammonemia. Hyperammonemia is also seen in nonketotic
hyperglycinemia, and mild hyperammonemia may occur in systemic
carnitine deficiency.
Measurement of Blood Ammonia
There are three satisfactory ways
of measuring blood ammonia: 1)
resin absorption methods, 2) enzymatic methods, and 3) methods
using ammonia electrodes. Diffusion methods, still in use in some
laboratories, generally yield high
values and therefore are not
recommended.
Resin absorption
methods involve the separation of ammonia
from plasma by a cation-exchange
resin. 36,37 Ammonia is then eluted
from the resin w i t h NaCI and
measured with the phenol-hypochlorite colorimetric reaction. The
resin step is necessary to separate
ammonia from plasma proteins
and other compounds that react
with phenol-hypochlorite. A commercial kit offered by Hyland
Diagnostics (Costa Mesa, CA)
yields satisfactory results, and the
method can be scaled d o w n to
require only 0.25 ml of plasma.
Enzymatic methods use glutamate dehydrogenase, 38 an enzyme
that converts alpha-ketoglutarate
and ammonia to glutamate while
converting NADH to N A D + . The
decrease in absorption of NADH
at 340 nm is usually measured;
however, the change in NADH can
also be measured fluorometrically, 39
or 1-C H alpha-ketoglutarate conversion to glutamate can be
measured. 40 Those methods that
measure changes in NADH must
avoid any changes in NADH due to
endogenous reactions—such as
conversion of pyruvate to lactate
by lactate d e h y d r o g e n a s e — b y
precipitating proteins with perchloric acid or by preincubation of
plasma until endogenous reactions
have gone to c o m p l e t i o n . A recent
report indicates that this problem
can also be overcome by using
NADPH instead of NADH. 4 1 4 2
Ammonia
electrode
methods
measure the change in the electrical potential as ammonia gas
(NH 3 ) diffuses across a semipermeable membrane and comes
into equilibrum with ammonium
ions (NH 4 + ). 4 3 The ammonia must
be measured at a pH such that
most of it is in a gaseous state. If
the pH is not carefully controlled,
labile amines in plasma will break
d o w n , increasing the measured
ammonia.
Diffusion
techniques
require
alkalinization of plasma so that
ammonia gas will diffuse into an
acidic trap. The trapped ammonia
is measured by titration or colorimetrically with phenol-hypochlorite. All diffusion methods yield
high values because the high pH
liberates ammonia f r o m labile
amines.
PhysioJogic Factors Affecting
Ammonia Levels
Arterial vs. venous blood. Some
laboratories always measure arterial blood ammonia levels; however, there is normally very little
difference between arterial and
venous blood ammonia levels. 44
Measurement of venous blood
ammonia is satisfactory for clinical
purposes.
Fasting vs. fed patients.
Even
after a high-protein meal, blood
ammonia levels increase very little
in normal individuals. However,
substantial increases may occur in
patients with impaired ammonia
metabolism.
Plasma vs. whole blood. Red cell
ammonia levels are from 50% to
100% higher than plasma ammonia levels.
Exercise. Moderate exercise has
little effect on blood ammonia, 4 4
but extreme exercise can cause a
marked elevation.
Technical Problems Affecting
Ammonia Determinations
Measurement of ammonia levels
in plasma is very difficult. The
c o n c e n t r a t i o n of a m m o n i a in
plasma is very low compared with
the potential ammonia that may be
released from labile compounds
in plasma and to the amount of
ammonia in the laboratory environment. While many modern
assays measure analytes present
in much lower concentrations,
few of these analytes are as ubiquitous as ammonia.
It is difficult to identify exactly
which precautions are necessary
to avoid contamination of specimens with ammonia. For example,
several investigators report success
in storing samples for short periods.
However, we have had difficulty
in storing specimens w i t h o u t en-
countering an increase in the ammonia level. The f o l l o w i n g precautions taken from our experience
and the experience of others 45
indicate the care that must be
taken in order to measure accurately plasma ammonia levels.
Patients should relax before the
test and should not smoke. Blood
should be drawn by accurate swift
venipuncture w i t h minimal turbulence and with minimal use of a
tourniquet (brief use of a tourniquet for ten to 15 seconds is
acceptable). The blood should be
drawn directly into a c o o l e d ,
heparinized, evacuated tube or it
should be immediately transferred
from a syringe into an o p e n , c o l d ,
heparinized tube w i t h o u t using a
needle. The tube should be immersed immediately in ice and
rotated to facilitate mixing and
cooling. The specimen should be
centrifuged rapidly at 4°C for
about five minutes. The plasma
should be separated as soon as
possible.
An experienced technologist
should be ready to perform the
assay immediately. The laboratory
should be kept as free of ammonia
as possible. Tobacco smoke, urine,
N H 4 O H and other potential ammonia contaminates should be excluded from the laboratory at all
times. Water and reagents must be
checked frequently to make sure
that they are free of ammonia and
formaldehyde; formaldehyde interferes w i t h the phenol-hypochlorite ammonia reaction. Glassware must be specially cleaned
and stored in very dilute sodium
hydroxide or sodium hypochlorite
and washed w i t h ammonia-free
water just before use. Assay tubes
should be kept covered at all times.
A procedure that avoids acidifying
or alkalinizing the sample should
be used.
If values obtained in a normal
individual are below 40 jumol/L,
LABORATORY MEDICINE • VOL 12, NO. 3, MARCH 1981 1 5 5
this indicates that precautions are
adequate.
Clinical Indications for
Measurement of Plasma Ammonia
Because of the increasing recognition of Reye's syndrome, it has
become standard practice to measure plasma ammonia in any child
with an unexplained encephalopathy. Such testing has contributed
to the increased identification of
patients with hyperammonemia
due to other causes. In adults, the
measurement of plasma ammonia
is often l i m i t e d , perhaps erroneously, to patients w i t h other
evidence of liver disease.
Measurement of plasma ammonia in encephalopathic patients
w i t h o u t obvious liver disease is
w o r t h w h i l e because it may lead to
an unsuspected diagnosis. The
value of plasma ammonia measurements in patients with severe generalized liver disease is more
limited, because there is relatively
little correlation between plasma
ammonia levels and encephalopathy.
In selected patients with mental
retardation, measurement of ammonia may be indicated to screen
for urea cycle disorders. Such
screening can probably be limited
to patients with episodic vomiting
or encephalopathy and to those
with a history of avoidance of highprotein foods. It should be kept in
mind that patients with mild urea
cycle disorders are not consistently
hyperammonemic.
In patients with hyperammonemia w h o d o not have generalized
liver disease, the level of plasma
ammonia is the prime determinant
of the results of treatment. Thus,
repeated measurements may be
very important. O n the other
hand, in patients with generalized
liver disease, including patients
with Reye's syndrome, following
156
the plasma ammonia level is less
important in evaluating therapy.
Alternate Tests for Assessing
Ammonia Metabolism
Because of the technical difficulties encountered in measuring
plasma ammonia, other methods
of assessing ammonia metabolism
have been considered. Spinal
fluid glutamine is elevated in
almost all patients w i t h hyperammonemia, 46 ' 47 and it is stable and
relatively easy to assay. Most
patients w i t h e n c e p h a l o p a t h y
must undergo a spinal tap for
other reasons; therefore, obtaining spinal fluid is not a p r o b l e m .
However, the need for spinal fluid
limits the use of this test in
patients w h o do not otherwise
need a lumbar puncture. O u r
institution offers plasma ammonia
determinations during the day but
only spinal fluid glutamine determinations at night.
Plasma glutamine and alanine
are also usually elevated in patients w i t h h y p e r a m m o n e m i a .
Measurement of these amino acids
may be a useful adjunct in some
situations; however, these amino
acid levels do not parallel plasma
ammonia levels closely enough to
be a substitute for plasma ammonia
measurement. Plasma alpha-ketoglutarate is inversely correlated
with blood ammonia. A recent
report 48 indicates that alpha-ketoglutarate may be sensitive to
changes in ammonia metabolism;
however, there has not been
enough experience w i t h measuring this c o m p o u n d to assess fully
its role in evaluating hyperammonemia.
Urinary orotic acid is elevated in
OTC deficiency. Our experience
with measurement of urinary orotic
acid indicates that it may be more
sensitive than measurement of
plasma ammonia in patients with
this disorder (unpublished observation).
LABORATORY MEDICINE • VOL. 12, NO. 3, MARCH 1981
Summary
Hyperammonemia may occur in
a number of disorders. In patients
with severe generalized liver disease, hyperammonemia is only a
part of the complex metabolic abnormalities. In other patients, such
as those with urea cycle disorders,
the hyperammonemia may be the
sole cause of the symptoms.
Several satisfactory methods are
available for measuring plasma ammonia, but special care is necessary to avoid specimen contamination from labile amines in plasma
and environmental ammonia.
References
1. Bessman, S.P., and Bessman, A.N., 1955. The
cerebral and peripheral uptake of ammonia
in liver disease with a hypothesis for the
mechanism of hepatic coma. J. Clin. Invest.
34:622-628.
2. Shorey, J., McCandless, D.W., and Schenker,
S., 1967. Cerebral alpha ketoglutarate in ammonia intoxication. Gastroenterology 53:706711.
3. Schenker, S., et al., 1967. Studies on the intracerebral toxicity of ammonia. J. Clin. Invest. 46:838-848.
4. Vergara, F, Plum, F., and Duffy, T. E., 1974.
Alpha ketoglutarate: Increased concentrations
in cerebrospinal fluid of patients in hepatic
coma. Science 183:81-83.
5. Benzi, G., et al., 1977. Metabolism and cerebral energy state: Effect of acute hyperammonemia in beagle dog. Biochem. Pharmacol. 26:2397-2404.
6. Chan, H., et al., 1977. Prolonged coma and
isoelectric electroencephalogram in child
with lysinuric protein intolerance. J. Pediatr.
91:79-81.
7. Batshaw, ML., et al., 1980. Cerebral dysfunction in asymptomatic carriers of ornithine
transcarbamylase deficiency. N. Engl. J. Med.
302:482-485
8. Walser, M., and Bodenlos, L.J., 1959. Urea
metabolism in man. J. Clin. Invest. 38:16171626.
9. Saheki.T., Katsunuma, T, andSase, M., 1977.
Regulation of urea synthesis in rat liver. J.
Biochem. 82:551-558.
10. Gamble, J.G., and Lehninger, A.L, 1973.
Transport of ornithine and citrulline across
the mitochondrial membrane. J. Biol. Chem.
248:610-618.
11. Valle, D., et al., 1979. Gyrate atrophy of
choroid and retina: Correction of hyperornithinemia by diet. Pediatr. Res. 13:483.
12. Samtoy, B., and Debeukelaer, M.M., 1980.
Ammonia encephalopathy secondary to urinary tract infection with Proteus mirabilis.
Pediatrics 65:294-296.
13. Snodgrass, P.J., and Delong, G.R., 1976.
Urea-cycle enzyme deficiencies and an increased nitrogen load producing hyperam-
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
monemia in Reye's syndrome. N. Engl. J. Med.
294:855.
Parkes, J.D., Sharpstone, P., and Williams, R.,
1970. Levodopa in hepatic coma. Lancet ii:
1341-1343.
Fischer, J.E., 1976. L-Dopa in hepatic coma.
Ann. Surg. 183:386-391.
Phillips, G.B., et al., 1952. The syndrome of
impending hepatic coma in patients with cirrhosis of the liver given certain nitrogenous
substances. N. Engl. J. Med. 247:238-246.
Gabuzda, G.J., Phillips, G.B., and Davidson,
C.S., 1952. Reversible toxic manifestations in
patients with cirrhosis of the liver given cationexchange resins. N. Engl. J. Med. 246:124130.
James, J.H., et al., 1979. Hyperammonemia,
plasma amino acid imbalance and blood-brain
amino acid transport: A unified theory of portalsystemic encephalopathy. Lancet ii:772-775.
Glasgow, A.M., Cotton, R.B., and Dhiensiri, K.
1972. Reyes syndrome: Blood ammonia and
consideration ofthe non-histologic diagnosis.
Am. J. Dis. Child. 124:827-836.
Gelehrter, T.D., and Snodgrass, P.H., 1974.
Lethal neonatal deficiency of carbamyl phosphate synthetase. N. Engl. J. Med. 290:430433.
Batshaw, M., Brusilow, S., and Walser, M.,
1975. Treatment of carbamyl phosphate
synthetase deficiency with keto analogues of
essential amino acids. N. Engl. J. Med. 292:
1085.
Short, LM., etal., 1973. Evidence for X-linked
dominant inheritance of ornithine transcarbamylase deficiency. N. Engl. J. Med. 288:7-12.
Campbell, A.G.M., et al., 1973. Ornithine
transcarbamylase deficiency: A cause of
lethal neonatal hyperammonemia in males.
N. Engl. J. Med. 288:1-6.
Levin, B., Oberholzer, V.G., and Sinclair, L,
1969. Biochemical investigations of hyperammonemia. Lancet ii: 170—174.
Glasgow, A.M., Kraegel, J.H., and Schulman,
J.D., 1978. Studies of the cause and treatment of hyperammonemia in females with
ornithine transcarbamylase deficiency. Pediatrics 62:30-37.
Goldstein, A.S., et al., 1974. Metabolic and
genetic studies of a family with ornithine
transcarbamylase deficiency. Pediatr. Res. 8:
5-12.
Whelan, D.T., Brusso, T., and Spate, M., 1976.
Citrullinemia: Phenotype variations. Pediatrics
57:935-941.
Brusilow, S.W., and Batshaw, ML., 1979. Arginine therapy of argininosuccinase deficiency. Lancet ii:124-125.
Synderman, S.E., et al., 1977. Argininemia. J.
Pediatr. 90:563-568.
Terheggen, H.G., etal., 1975. Familial hyperargininaemia. Arch. Dis. Child. 50:57-62.
Simell, O., et al., 1975. Lysinuric protein
intolerance. Am. Med. 59:229-240.
Simell, O., 1975. Diamino acid transport into
granulocytes and liver slices of patients with
lysinuric protein intolerance. Pediatr. Res. 9:
504-508.
33. Fell, V., 1974. Ornithinemia, hyperammonemia,
and homocitrullinuria. Am. J. Dis. Child. 127:
752-756.
34. Batshaw, M.L., and Brusilow, S.W., 1978.
Asymptomatic hyperammonemia in low birthweight infants. Pediatr. Res. 12:221-224.
35. Ballard, R.A., et al., 1978. Transient hyperammonemia of the preterm infant. N. Engl. J.
Med. 299:920-925.
36. Oberholzer, V.G., et al., 1976. Microscale
modification of a cation-exchange column
procedure of plasma ammonia. Clin. Chem.
22:1976-1981.
37. Wu, J., Ash, K.O., and Mao, E., 1978. Modified
micro-scale enzymatic method for plasma
ammonia in newborn and pediatric patients:
Comparison with a modified cation-exchange
procedure. Clin. Chem. 24:2172-2175.
38. Doumas, B.T., 1979. Performance of the Du
Pont aca ammonia method. Clin. Chem. 25:
175-178.
39. Nazar, B.L, and Schoolwerth, A.C., 1979. An
improved microfluorometric enzymatic assay
for the determination of ammonia. Anal. Biochem. 95:507-511.
40. Kalb, V.F., Jr., etal., 1978. A new and specific
assay for ammonia and glutamine sensitive to
100 pmol. Anal. Biochem. 90:47-57.
41. Kli, P., and Shull, B.C., 1979. Fixed-time kinetic assay of plasma ammonia, with NADPH
as factor, with a centrifugal analyzer. Clin.
Chem. 25:611-613.
42. Humphries, B.A., et al., 1979. Automated enzymatic assay for plasma ammonia. Clin.
Chem. 25:26-30.
43. Attili, A.F., Autizi, D., and Capocaccia, L,
1975. Rapid determination of plasma ammonia using an ion-specific electrode. Biochem. Med. 14:109-116.
44. Dawson, A.M., 1978. Regulation of blood
ammonia. Gut 19:504-509.
45. Gerron, G.G., et al., 1976. Technical pitfalls
in measurement of venous plasma NH3 concentration. Clin. Chem. 22:663-666.
46. Hourani, B.T., Hamlin, E.M., and Reynolds,
T.B., 1971. Cerebrospinal fluid glutamine as
a measure of hepatic encephalopathy. Arch.
Intern. Med. 127:1033-1036.
47. Glasgow, A.M., and Dhiensiri, K., 1974. Improved assay for spinal fluid glutamine and
values of children with Reye's syndrome.
Clin. Chem. 20:642-644.
48. Batshaw, ML., and Brusilow, S.W., 1980.
Alpha ketoglutarate as a harbinger of hyperammonemia in urea cycle enzymopathies.
Pediatr. Res. 14:519.
•
Review Questions
Chemistry III
1. It is postulated that the underlying mechanism of ammonia neurotoxicity is a shift in reaction equilibrium caused by an elevated
neuronal ammonia. What compounds are involved in this reaction
and what is the physiologic effect?
2. An ammonium ion is presented to a hepatocyte. Trace the possible
fate of this ion and the end products that may be formed.
3. A patient's plasma ammonia level is noted to rise during hyperalimentation. What is a possible explanation?
4. Male infants with ornithine transcarbamylase (OTC) deficiency
usually die; females usually live, but with varying degrees of
mental retardation. Explain why.
5. A physician indicates that an elevated plasma ammonia level
reported by the laboratory is too high to be consistent with the
patient's clinical condition. What sources of laboratory error
should be considered?
6. Give three clinical indications for measuring plasma ammonia.
7. Evaluate the following alternate laboratory determinations for the
diagnosis of hyperammonemia:
a. Spinal fluid glutamine
b. Plasma alanine
c. Urinary orotic acid
Self-assessment exam order form can be found on page 175.
LABORATORY M E D I C I N E • VOL. 12, NO. 3, MARCH 1981
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