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Clinical Chemistry 45: 1255-1260, 1999;
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(Clinical Chemistry. 1999;45:1255-1260.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Determination of the Sum of Bilirubin Sugar Conjugates in Plasma by Bilirubin Oxidase

Basil T. Doumas1,a, Fred Yein2, Billy Perry1, Bernadine Jendrzejczak1 and Arthur Kessner2

1 Medical College of Wisconsin, Department of Pathology, P.O. Box 26509, Milwaukee, WI 53226-0509.

2 Beckman Instruments Inc., 200 South Kraemer Blvd., Brea, CA 92822-8000.
a Author for correspondence. Fax 414-456-6305; e-mail bdoumas{at}mcw.edu

Background: A reliable indicator of cholestasis is the presence of abnormal concentrations of bilirubin mono- and diglucuronide [conjugated bilirubin (CB)] in blood. A routine assay of CB is available only to those who possess a certain type of clinical analyzer. We describe a two-point manual method for CB that could be adapted as a rate assay to automated clinical analyzers.

Methods: The measurement of CB is based on its oxidation to biliverdin by bilirubin oxidase. The resulting decrease in absorbance at 460 nm is proportional to the CB concentration. The assay is calibrated with solutions of ditaurobilirubin in human serum.

Results: Under the conditions of the assay (0.1 mol/L glycine buffer, pH 10.0; reaction time, 2 min), only 5% of unconjugated bilirubin is oxidized and {delta}-bilirubin is not oxidized at all. Results obtained with the bilirubin oxidase method agreed well with those obtained by HPLC. The long-term CVs at CB concentrations of 6 and 63.4 mg/L were 20% and 2.6%, respectively. The reference values, established by analyzing 51 plasma specimens from healthy adults, were 0.0–1.2 mg/L, with a mean value of 0.2 mg/L.

Conclusions: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measurement of CB in blood is superior to the measurement of direct bilirubin because an abnormal concentration of direct bilirubin does not necessarily indicate the presence of cholestasis.© 1999 American Association for Clinical Chemistry




The following articles in journals at HighWire Press have cited this article:


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 Appl. Environ. Microbiol.Home page
S.-i. Sakasegawa, H. Ishikawa, S. Imamura, H. Sakuraba, S. Goda, and T. Ohshima
Bilirubin Oxidase Activity of Bacillus subtilis CotA
Appl. Envir. Microbiol., January 1, 2006; 72(1): 972 - 975.
[Abstract] [Full Text] [PDF]


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