Ammonia

Determination

By:
Alanano, John Paul T.
BSMT-3a
 AMMONIA:
Ammonia is formed in the
deamination of amino acids
during protein metabolism.It is
removed from the circulation and
converted to urea in the liver.
Free ammonia is toxic; however,
ammonia is present in the plasma
in low concentrations.
 Sample
• Arterial blood is the preferred specimen for
measurement of ammonia. Although venous blood is
not recommended, if used, tourniquets should be used
minimally, Specimens should be kept in ice water until
separation of cells from plasma occurs
• Heparin and EDTA are suitable
anticoagulants. Commercial collection
containers should be evaluated for ammonia
interference before a new lot is put into use.

• Specimens should be assayed as

soon as possible or frozen.
• Hemolysis should be avoided.
• Frozen plasma is stable for
several days at 20°C.
• Cigarette smoking by the patient is
a significant source of ammonia
contamination. It is recommended
that patients do not smoke for
several hours before the sample is
collected.
•Centrifuge for a minimum of 10
minutes. (cold). Specimen must
be processed cold. Place
specimen on ice and assay
immediately.
• Taking any medicines. Because many medicines
can interfere with test results. Your doctor may instruct
you to stop taking certain medicines for several days
before having an ammonia test.

• Other sources of contamination

include urine and ammonia in
detergents, glassware, reagents,
and water.
• Prolonged application of a tourniquet
or fist-clenching while obtaining the
blood sample or improper specimen
handling can result in a falsely elevated
blood ammonia level.
Two approaches have been
used for the measurement of
plasma ammonia.
 One is a two-step approach in which ammonia
is isolated from the sample and then assayed. The
second involves direct measurement of ammonia by
an enzymatic method or ion-selective electrode.
 One of the first analytic methods for ammonia, developed
by Conway in 1935, the technique employs a special
microdiffusion dish, this consist of a shallow circular glass
vessel (40-70mm in diameter) containing a central well and w/
a closely fitting lid sealed w/ grease, the lid closes the outer
annular space but does not touch the wall of the central wall.
The sample is placed in the outer annalus and a gas-absorbing
reagent is placed in the centre well. The specific reagent is
added to the sample, the lid is closed and diffusion of evolve
gas allowed to proceed, subsequently the remaining gas-
absorbing is titrated,
This method is convenient and the most common technique
used currently. Ammonia is typically measured by enzymatic
assays using glutamate dehydrogenase, which catalyzes the
reaction of α-ketoglutarate and ammonia to form glutamate,
with oxidation of NADPH to NADP as the indicator.
NADPH is the preferred coenzyme because it is used
specifically by glutamate dehydrogenase. This method is used
on many automated systems and is available as a prepared kit
from numerous manufacturers.
 Dry Slide Method
 Ammonia is also measured via a
dry slide method using alkaline pH
buffers to convert all ammonium ions
to ammonia gas, with bromphenol
blue as the indicator.
In this method, ammonia reacts with
an indicator to produce a colored
compound that is detected
spectrophotometrically. Direct
measurement using an ion selective
electrode has been developed. The
electrode measures the change in pH
of a solution of ammonium chloride
as ammonia diffuses across a
semipermeable
membrane.
~ END ~