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Thiobarbiturates interfere with the Dade Behring aca ammonia test.

To the Editor:

A 13-month-old boy with myoclonic epilepsy gave abnormal results for ammonia concentration in EDTA plasma and urine on an automatic clinical analyzer (aca; Dade Behring), which printed out negative concentration values with an error message. The assay reaction mixture in the aca pack showed no turbidity or precipitates, and there was no clue for the explanation of these results at that time. Approximately 6 weeks later, a 21-month-old boy with heart disease also gave negative ammonia concentrations. Both had received injections of sodium thiopental solution for prevention of convulsions, but no other drug had been given to both boys in common. The concentration of thiopental, a thiobarbiturate, in the plasma of the first boy was 135 mg/L by an HPLC-ultraviolet detection method.

This assay interference could be reproduced with thiopental added to control plasma and also with thiamylal (another thiobarbiturate) added to control plasma. Two barbiturates, pentobarbital and phenobarbital, gave no interference when added to control plasma.

Various amounts of thiopental were added to plasma specimens for the dose-response study. As the result for one specimen shows (Table 1), the ammonia concentration measured by the aca test was inversely proportional to the thiopental concentration. The clinically effective concentration of thiopental in plasma for anesthesia is 25-80 mg/L according to a manufacturer of this drug (Tanabe Pharmaceutical Company). This concentration range would produce a decrease in the ammonia concentration of 20-64 [micro]mol/L. Because the reference interval for ammonia is 11-35 [micro]mol/L, plasma samples from patients receiving thiopental would give anomalously decreased or even negative ammonia values as seen in the boys described above.

The ammonia test is based on the following reaction of glutamate dehydrogenase: N[H.sub.4.sup.+] + [alpha]-ketoglutarate + NADPH [right arrow] L-glutamate + NAD[P.sup.+] + [H.sub.2]O. The analyzer measures the decreasing absorbance of NADPH at 340 nm. Another ammonia test kit, Determiner[R] [NH.sub.3] (Kyowa Medix), based on the same enzyme reaction, was implemented on a Hitachi 717 automatic analyzer (Hitachi). To our surprise, no interference was observed with this test (Table 1). Following are the differences between the aca and Determiner methods: (a) source of glutamate dehydrogenase, bovine liver vs yeast; (b) pH of assay buffer, pH 7.4 vs pH 8.25; (c) additives, e.g., surfactant, pH buffer salt, and chelating reagents, although detailed descriptions are not given for these ingredients; (d) absorbance measurement mode, single wavelength of 340 nm vs double wavelength of 340 nm (primary) and 405 nm (secondary); (e) reaction monitoring method, rate assay vs end-point assay.

According to Dade Behring Japan Ltd., there had been no reports on ammonia assay interference by thiobarbiturates. The mechanism of this interference is not yet clear. No reports have been found on the interaction of thiobarbiturates with this enzyme. Another possibility is that unknown extra reactions occur and counteract the decrease in NADPH absorbance or even produce NADPH from traces of [NADP.sup.+] in the reaction vessel.

Yoshiyuki Fujimura (1)

Takashi Nishikawa (2,3) *

Hideki Ohtani (1,2)

(1) Department of Clinical Laboratory

Kitasato University Hospital

1-15-1, Kitasato, Sagamihara

Kanagawa 228-8555, Japan

(2) Department of Clinical Pathology

Kitasato University School of Medicine

1-15-1, Kitasato, Sagamihara

Kanagawa 228-8555, Japan

(3) Kitasato Laboratory

Sumitomo Metal Bio-science Inc.

1-15-1, Kitasato, Sagamihara

* Address correspondence to this author at: Department of Clinical Pathology, Kitasato University School of Medicine, 1-15-1, Kitasato, Sagamihara, Kanagawa 228-8555, Japan. Fax 81-42778-4340; e-mail
Table 1. Apparent ammonia concentrations in plasma
with added thiopental.

 Ammonia concentration
 in plasma, [micro]mol/L
Sodium thiopental
in plasma, mg/L aca Determiner
0 46.5 42.9
20 29.4 41.8
60 2.94 41.2
100 -17.1 41.8
200 -65.3 40.6

*Address correspondence to this author at: Department
of Clinical Pathology, Kitasato University School of
Medicine, 1-15-1, Kitasato, Sagamihara, Kanagawa 228-8555,
Japan. Fax 81-427-78-4340; e-mail
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Title Annotation:Letters
Author:Miura, Yoshinori; Fujimura, Yoshiyuki; Nishikawa, Takashi; Ohtani, Hideki
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:May 1, 2000
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