A Fluorescence-based Quantitative Analysis for Total Bilirubin in Blood and Urine

Tran Tien Tai, MD, PhD; Yasushi Adachi, MD, PhD; Shigeru Taketani, PhD


Lab Med. 2022;53(1):6-11. 

In This Article

Abstract and Introduction


Background: Bilirubin is a catabolic product of heme metabolism that circulates in the bloodstream in its unconjugated or glucuronide-conjugated form. Because the accumulation of bilirubin in the blood is a common symptom of liver diseases, its measurement in plasma (serum) is important for the diagnosis of these diseases.

Method: We developed a method to assess total bilirubin levels in serum and urine, using the fluorescent protein UnaG and β-glucuronidase.

Results: Our results indicate good correlation in serum total bilirubin levels between UnaG and the conventional bilirubin oxidase (BOD) methods. We found low levels of conjugated and unconjugated bilirubin in the urine of healthy subject individuals. Urinary bilirubin levels were elevated in patients with liver or bile duct diseases. A simple spot test of bilirubin using serum and urine showed a strong signal in patients with liver diseases.

Conclusion: The proposed method to assess bilirubin levels in serum and urine will contribute to the accurate diagnosis of health conditions such as jaundice, anemia, and liver disease.


Bilirubin is a yellow pigment and the catabolic product of heme metabolism, which is formed by the breakdown of heme in hemoglobin, myoglobin, cytochromes, catalase, peroxidase, and tryptophan pyrrolase.[1] Bilirubin circulates in the bloodstream in its unconjugated insoluble form (indirect bilirubin) or glucuronide-conjugated soluble form (direct bilirubin).[2,3] Serum bilirubin is mostly unconjugated and is tightly bound to serum albumin. It is taken up by hepatocytes and is conjugated with glucuronic acid by the enzyme β-glucuronyltransferase, producing conjugated bilirubin. This process is clinically important because bilirubin accumulates in body tissues and is elevated in the blood of most patients with jaundice. The accumulation of this substance in the blood is a common symptom of liver diseases.[3,4] Thus, bilirubin in plasma (serum) has potential as a marker of these diseases. The accurate measurement of blood bilirubin levels is also important, particularly in the treatment of neonatal jaundice, because the decision to initiate or discontinue treatment is often based on bilirubin levels.[3–5]

Most bilirubin is secreted into bile and delivered into the small intestine. After several modifications, the metabolite, urobilinogen (or urobilin) is excreted by the kidneys into urine.[5,6] Normal urine contains as much as 4 mg urobilinogen per day; excessive amounts of urobilinogen in urine indicate hepatic damage and increased red blood cell (RBC) destruction. Accompanied by an increase in excreted urobilinogen, bilirubin levels in urine may be elevated by several diseases, including hemolysis, as well as hepatic and renal dysfunctions.[7] However, because individual laboratories establish their own normal reference values and the data obtained are dependent on the method performed, this is not a reliable index of diseases.[8–10] Thus, a suitable method for measuring urinary bilirubin has not yet been established.

The eel fluorescent protein UnaG binds with high affinity to bilirubin;[11,12] thus, the quantification of bilirubin in tissues and serum by the reaction of UnaG with heme oxygenase has been suggested as a useful application. We previously reported a simple and sensitive assay of heme oxygenase activity.[13] Other investigators[14,15] also demonstrated the use of UnaG to measure unconjugated bilirubin in serum. We have continued our research on the availability of UnaG for bilirubin assays and herein describe a simple method to measure total bilirubin levels in serum using only a small amount of serum (<1 μL) and UnaG. Urinary bilirubin levels markedly differed between patients with liver diseases and healthy subjects.