Analysis of Bilirubin levels in serum using colorimetry

Bilirubin is a product of the breakdown of heme group, a component found in hemoglobin (Hb), present in red blood cells, as well as in other hemoproteins such as myoglobin, cytochromes, and catalase. When red blood cells reach senescence, they break down, releasing Hb into the bloodstream. 

Subsequently, the heme group of this Hb oxidizes and transforms into biliverdin, which is then reduced to form free bilirubin. 

Finally, the free bilirubin conjugates with albumin and travels to the liver, where it is processed and ultimately excreted into the gallbladder. It remains stored there until needed for the digestion process.

Bilirubin Concentration

Under normal conditions, the highest concentration of bilirubin is found in the gallbladder, followed by the liver and finally the plasma. 

Elevated levels of bilirubin in plasma (hyperbilirubinemia) may indicate a disruption in the liver’s bilirubin metabolism, correlating with various liver and hemolytic diseases. Therefore, assessing the level of bilirubin in serum is routinely performed in medical exams to check the liver’s functionality.

Colorimetric Method

There are various methods for assessing bilirubin in serum, with the colorimetric method being the most common. 

Briefly, this method uses a reagent, diazotized sulfanilic acid, which transforms bilirubin into colored azobilirubin, allowing detection with a spectrophotometer. Thus, the intensity of the color formed is proportional to the concentration of bilirubin in the sample.

However, before carrying out this protocol, it is necessary to isolate the serum from the patient’s blood sample. For this purpose, a blood sample is taken in a 5 mL non-anticoagulant blood collection tube and centrifuged at 1,500 G for 15 minutes at room temperature. The Digicen 22 R is the best option for serum isolation due the temperature control that it achieves. Additionally, it features our REI System, which allows for quick and secure installation and locking of the rotor without the need for tools.

After centrifugation, the sample is divided into two fractions. The upper fraction, clear and transparent in appearance, corresponds to the blood serum, which is transferred to another tube to begin the bilirubin assessment protocol.

The assessment of bilirubin in serum is routinely conducted in clinics and holds special importance in patients with liver disease. It is generally performed using a colorimetric method, preceded by the isolation of the patient’s serum through a centrifugation step.