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Urine concentration of biopyrrins: a new marker for oxidative stress in vivo.

To the Editor:

Currently interest in reactive oxygen species (ROSS) is heightened because of their involvement in cardiovascular, neurodegenerative, and chronic inflammatory diseases and cancer. The high reactivity of ROSS coupled with their very short life span is a stumbling block in the direct measurement of these species in human subjects (1).

Bilirubin, a metabolite of heme, apparently functions as an antioxidant in vivo by reacting with ROSS and, as a result, becomes oxidized (2). Hence, the oxidative metabolites of bilirubin could be a biological marker for in vivo ROS production. Recently Yamaguchi et al. (3) identified novel oxidative metabolites of bilirubin in human urine as substances that are recognized by an anti-bilirubin monoclonal antibody, 24G7, but are negative in the diazo reaction. These metabolites are tripyr-role biocompounds and are designated biopyrrins. The production of biopyrrins increases in oxidatively insulted rats subjected to endotoxin treatment (4) and ischemia-reperfusion (5). Increased excretion ofbiopyrrins in urine has been reported in patients who have undergone resection of esophageal cancer (6). Thus, the production of biopyrrins appears to reflect the degree of oxidative stress in vivo, and the measurement of biopyrrins may be useful for estimating oxidative stress. To date, however, the production and excretion of biopyrrins in healthy human subjects have not been critically examined.

The first urine in the morning was collected from 33 healthy volunteers (33 [+ or -] 18 years old) and from 14 patients (56 [+ or -] 13 years old) who had undergone abdominal operations lasting >5 h. Subjects gave informed consent according to the ethics rules of Kyushu University Hospital. The biopyrrin concentration in urine was corrected by the concentration of creatinine in the same sample. The samples from healthy subjects were collected periodically, 5 to 25 times per person (average, 18), for 1 month. The total number of samples collected was 600. For each individual, the mean [(M.sub.i)] of the urine concentration of biopyrrins and its standard deviation [(SD.sub.i)] were determined. The [CV.sub.i] (defined as [SD.sub.i]/[M.sub.i)] was used to assess day-to-day variation, i.e., the degree of change of the biopyrrin concentration between days in a given individual. Subsequently, a mean, SD, and CV were calculated for each group of the healthy subjects.

The biopyrrin concentration was determined by subtracting the concentration of the diazo reaction-positive substances (i.e., bilirubin) from the concentration of the anti-bilirubin monoclonal antibody 24G7-reactive substances. The concentration of the anti-bilirubin monoclonal antibody 24G7-reactive substances was measured with a biopyrrin measuring kit (Shino-test), using bilirubin as a calibrator according to the manufacturer's instructions.

The concentrations ([+ or -] SD) of biopyrrins in healthy male and female subjects and in the total number of healthy subjects were 0.82 [+ or -] 0.29, 0.74 [+ or -] 0.11, and 0.79 [+ or -] 0.24 [micro]mol per gram of creatinine, respectively. The mean values of the day-to-day CV and SDs of the means in healthy male and female subjects and in the total number of healthy subjects were 0.33 [+ or -] 0.08, 0.32 [+ or -] 0.06, and 0.33 [+ or -] 0.08, respectively. The physiological change of the urine concentration of biopyrrins between days was small, and the concentration seldom fluctuated more than twice the mean value in a given individual.

The concentration of biopyrrins increased markedly from 0.74 [+ or -] 0.44 [micro]mol/g creatinine before the operation to 3.37 [+ or -] 6.24, 3.66 [+ or -] 6.88, and 2.59 [+ or -] 2.71 [micro]mol/g creatinine at 1, 3, and 5 days after the operation, respectively. The value decreased to 0.83 [+ or -] 0.61 [micro]mol per gram of creatinine at 14 days after the operation. The increase was not simply related to the increase in serum bilirubin because the concentrations of serum bilirubin and urine biopyrrins were not correlated significantly.

We conclude that biopyrrins may prove useful as a practical clinical test for the estimation of oxidative stress in vivo.

This work was supported in part by a Grant-in-aid for Scientific Research from the Ministry of Education, Sciences, Sports, and Culture of Japan. We thank Shino-test Corporation for generously providing a biopyrrin measuring kit. We extend special thanks to 5. Narayanan (New York Medical College, New York, NY) for critical reading.


(1.) Rumley AG, Patersin JR. Analytical aspects of antioxidants and free radical activity in clinical biochemistry. Ann Clin Biochem 1998;35:181-200.

(2.) Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science 1987;235:1043-6.

(3.) Yamaguchi T, Shioji I, Sugimoto A, Komoda Y, Nakajima H. Chemical structure of a new family of bile pigments from human urine. J Biochem 1994;116:298-303.

(4.) Yamaguchi T, Horio F, Hashizume T, Tanaka M, Ikeda S, Kakinuma A, et al. Bilirubin is oxidized in rats treated with endotoxin and acts as a physiological antioxidant synergistically with ascorbic acid in vivo. Biochem Biophys Res Commun 1995;214:11-9.

(5.) Yamaguchi T, Terakado M, Horio F, Aoki K, Tanaka M, Nakajima H. Role of bilirubin as an antioxidant in an ischemia-reperfusion of rat liver and induction of heme oxygenase. Biochem Biophys Res Commun 1996;223:129-35.

(6.) Tsujinaka T, Fujita J, Morimoto T, Shiozaki H, Monden M, Yamaguchi T, et al. Clinical significance of urinary excretion of bilirubin oxidative derivatives following resection of esophageal cancer: preliminary report. Jpn J Surg 1995;96: 495.

Kayoko Shimoharada [1] Sumiko Inoue [1] Mutsuko Nakahara [1] Naoto Kanzaki [2] Shuji Shimizu [2] Dongchon Kang [1] Naotaka Hamasaki [1] Sachiko Kinoshita [1] *

[1] Clinical Laboratory

Kyushu University Hospital

Fukuoka 812-8582, Japan

[2] Department of First Surgery

Kyushu University Faculty of Medicine

Fukuoka 812-8582, Japan

* Author for correspondence. Fax 51-92642-5772
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Title Annotation:Letters
Author:Shimoharada, Kayoko; Inoue, Sumiko; Nakahara, Mutsuko; Kanzaki, Naoto; Shimizu, Shuji; Kang, Dongcho
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Dec 1, 1998
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