College of American Pathologists
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  Q & A





cap today

September 2002

Q.  We perform blood ammonia determinations using plasma from venous blood. One of our gastroenterologists insists that arterial blood is the preferred specimen for accurate ammonia levels. I have seen several references to the use of this type of specimen in the gastroenterology literature, but I have been unable to find any recommendation for the routine use of arterial blood in clinical laboratory textbooks. Can you help me resolve this?

A.  Plasma ammonia determinations have always been problematic for a number of reasons. The normal concentration is in the micromolar range, making the test susceptible to a variety of preanalytical errors. Even when carefully measured, plasma ammonia concentrations have usually been found to correlate poorly with the clinical stage of hepatic encephalopathy whether arterial or venous blood is used, and although ammonia is usually elevated, a normal level does not rule out early-stage hepatic encephalopathy.

Ammonia is produced in the gastrointestinal tract by the action of bacterial enzymes on proteins and amino acids. It enters the portal circulation and is normally metabolized in the liver to urea and glutamine. When the liver is unable to perform this function, increased amounts of ammonia enter the arterial circulation and diffuse across the blood-brain barrier.

Ammonia levels in peripheral venous blood are affected by additional variables. Muscle cells produce ammonia during exercise, but can take up ammonia when resting. It has been shown that the arteriovenous difference is near zero in normal individuals at rest, but that in hyperammonemic states, muscle serves as an important site for uptake of ammonia with subsequent conversion to glutamine. Arteriovenous differences in chronic hepatic insufficiency are typically 20-30 ┬Ámol/L. 1,2 Venous ammonia will increase if a tourniquet is used during venipuncture. Because of these factors, arterial blood is frequently specified as the preferred specimen for ammonia determinations. 3

Another complication is that total ammonia in plasma exists as both NH 3 and as ammonium ion, NH 4 +. The neutral NH 3 molecule crosses the blood-brain barrier much more readily than the NH 4 + cation and so the NH 3 fraction is really the component of interest with respect to the development of encephalopathy. The pKa of NH 4 + at 37°C is 8.9, so at pH 7.4 plasma ammonia exists as 97 percent NH 4 + and three percent NH 3 . In alkalosis, which often accompanies hyperammonemia, the fraction of NH 3 increases significantly. At pH 7.6 it is 4.8 percent, an increase of 60 percent, and the correlation of plasma ammonia with hepatic encephalopathy is improved if pNH 3 (in arterial blood) is used rather than total ammonia concentration. 4

Nevertheless, it is unlikely that ammonia is solely responsible for the encephalopathy of hepatic insufficiency, and recent literature emphasizes the view that while elevated ammonia levels may be useful in suggesting a hepatic origin for an encephalopathy of unknown etiology, the test is not useful in patients with known liver disease. 5,6

In summary, measurement of the NH3 fraction of total ammonia in arterial plasma should maximize the sensitivity of this test for the diagnosis of hepatic encephalopathy, and the use of arterial blood should not be discouraged. On the other hand, if venous blood is collected from a patient at rest, without a tourniquet, and analyzed promptly, significant elevations in arterial ammonia should also be reflected in venous plasma, though to a lesser degree. Once a diagnosis of hepatic encephalopathy is made, the response to therapy should be monitored with criteria other than ammonia determinations.

1.  Ganda OP, Ruderman NP. Muscle nitrogen metabolism in chronic hepatic insufficiency. Metabolism. 1976;25: 427-435.
2.  Stahl J. Studies of the blood ammonia in liver disease. Ann Intern Med. 1963;58: 1-24.
3.  Lockwood AH. Hepatic Encephalopathy. Boston, Mass: Butterworth-Heinimann; 1992.
4.  Kramer L, Tribl G, Gendo A, et al. Partial pressure of ammonia versus ammonia in hepatic encephalopathy. Hepatology. 2000;31:30-34.
5.  Tolman KG, Rej R. Liver Function. In: Burtis CA and Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, Pa.: WB Saunders Co.; 1999.
6.  Wolf DC. Hepatic encephalopathy. eMedicine Journal. July 9, 2001. Available at: Accessed Aug. 22, 2002.

Robert W. Burnett, PhD
Department of Pathology
and Laboratory Medicine
Hartford Hospital
Hartford, Conn.

Consultant, CAP Chemistry Resource Committee

Q.  We have an oncologist who orders methotrexate levels immediately after a dose. We have to make high dilutions to get the result within an analytical range. It was my understanding that this drug should be monitored at approximately 24 and 48 hours postdosing. Is this incorrect?

A.  Methotrexate is used to treat a variety of malignancies. The list includes acute lymphoblastic leukemia, lymphoma, choriocarcinoma, osteogenic sarcoma, and carcinomas of the breast, lung, and head and neck. The drug may be administered orally or intravenously, and the dose depends on the type of cancer. High-dose therapy (> 50 mg/m 2 ) may be associated with significant toxicity. Therapeutic monitoring of high-dose metho-trexate is essential to guide the timing and amount of leucovorin (folinic acid) a patient should receive to "rescue" him or her from the drug's toxic effects.

The serum concentration of methotrexate is usually monitored at 24, 48, and, if necessary, 72 hours after a single dose. The toxic concentrations at each time point are as follows:

  • 24 hours          ≥5 µmol/L
  • 48 hours          ≥0.5 µmol/L
  • 72 hours          ≥0.05 µmol/L
The times at which the drug is monitored and the toxic concentrations may vary with different dosing regimens. Methotrexate is eliminated primarily through the kidneys; 70 to 90 percent of the drug is excreted unchanged. Renal disease, decreased urine flow, and some drugs reduce its clearance and raise the serum concentration. The clearance of methotrexate is also decreased in patients with ascites and pleural effusions, as the drug can collect in these fluid spaces.

When methotrexate is measured in serum immediately after a dose, the result naturally will be very high. However, this provides little or no information about the drug's subsequent metabolism and is not used as a guide to leucovorin rescue. Since the purpose of monitoring the drug is to assess toxicity and treat when indicated, I can see no value in monitoring the drug immediately after administration

William E. Schreiber, MD
Consultant pathologist
Department of Pathology
and Laboratory Medicine
Vancouver Hospital and
Health Sciences Center
Vancouver, British Columbia

Member, CAP Therapeutic Drug Monitoring/Endocrinology
Resource Committee