Q&A column

Editor: Frederick L. Kiechle, MD, PhD

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Q. What is the ideal collection tube for measuring the level of ammonia in blood? Is a tube containing EDTA suitable?

A.September 2021—Importance of measuring ammonia. Ammonia measurement is part of the differential diagnosis for hyperammonemia. Although ammonia is a normal by-product of amino acid metabolism, low plasma ammonia concentrations are caused by glutamine synthesis and urea synthesis in the liver.¹ An elevated level of ammonia suggests an abnormality in nitrogen homeostasis, most likely due to liver dysfunction.² Other possible causes of hyperammonemia include inborn errors of metabolism, gastric bypass, and drugs that have a direct effect on ammonia metabolism (e.g. valproic acid) or cause hepatotoxicity (e.g. acetaminophen).³

High concentrations of ammonia are toxic, especially to the central nervous system, and often manifest as encephalopathy, lethargy, vomiting, and seizures, among other symptoms.¹ When hyperammonemia is confirmed, prompt intervention is required to avoid long-term neurological damage. Further investigation is required to establish the cause of the condition.¹

Methods used to measure ammonia. Although several methods can be used to measure ammonia, the most common are an enzymatic method (direct) and a microdiffusion method (indirect).^1,4,5 The enzymatic approach consists of reducing ammonia and alpha-ketoglutarate to glutamate by glutamate dehydrogenase in the presence of NADH or NADPH. The decrease in concentration of NADH or NADPH can be detected as a change in absorbance using reflectance spectroscopy.

In the microdiffusion method, ammonium ions are converted to gaseous ammonia in an alkaline environment in which free ammonia passes through a semipermeable membrane to an indicator layer (e.g. bromophenol blue). The color change in the presence of ammonia can be detected spectrophotometrically.

Considerations in sample handling. Whenever plasma ammonia results are elevated, sampling artifacts should be taken into account. Preanalytical errors are a main confounding factor in interpreting ammonia results. The following are recommendations for handling blood samples used to measure ammonia.

• Collect blood in an ammonia-free environment, avoiding exposure to air and water or to tubes contaminated with ammonia.
• Promptly centrifuge the sample to separate plasma from cell components. A hemolyzed sample is unacceptable because red blood cells contain high concentrations of ammonium.
• Measure the ammonia concentration within 15 minutes of collecting a blood sample, keeping the sample on ice until it is analyzed. (Maintaining the sample on ice delays spontaneous ammonia formation.)
• Freeze the sample if it cannot be processed within 15 minutes.
• Repeat the blood ammonia measurement if sample-handling errors are suspected.

Blood collection tube selection. Common practice is to collect a blood sample in an ethylenediaminetetraacetic acid or heparin tube to measure blood ammonia. There is no general consensus on the superiority of EDTA versus heparin as an anticoagulant in blood collection for measuring this waste product.^5-7

Avoid using any tube that could be contaminated with ammonia. A serum sample in a tube with a clot activator (red top) is not acceptable because clotting increases ammonia production.⁸