3. Before the analysis: methods - Methods used for the measurement of ammonia, limitations and pitfalls

Ammonia can be determined in plasma by a variety of different approaches applying titration, colorimetric/fluorimetric, electrode-based, and enzymatic methods. In most routine labs, the reaction of glutamate dehydrogenase (GLDH) is used for ammonia determination.

GLDH catalyses the following reaction:

This reaction leads to a change in absorption at 340 nm which is the result of the oxidation of NADPH; this change in absorption is dependent on the initial ammonia concentration and therefore allows quantifying ammonia.

The advantage of this reaction is that it runs very fast making results available within only a few minutes.

For more details, please consult reference 6

A few pre-analytical aspects need to be kept in mind concerning ammonia determination:

1. If a tourniquet is used and the blood is no longer free flowing, hemolysis and contamination with tissue fluid might lead to false high ammonia.
2. The blood needs to be mixed with an anticoagulant to prevent hemolysis within the sample.
3. Postprandial ammonia levels are higher than fasting levels.
4. Physical exercise (even such as crying in an infant) might lead to falsely high ammonia.
5. For transport, the sample should be immediately cooled to avoid hemolysis within the sample.
6. The sample should be used straight away as storage may lead to hemolysis.

A full list of references for further reading can be found at the end of the course.