Hello, conjugated bilirubin is often incorrectly called "direct bilirubin" and unconjugated bilirubin is incorrectly called "indirect bilirubin". Direct and indirect refer solely to how compounds are measured or detected in solution. Direct bilirubin is any form of bilirubin which is water-soluble and is available in solution to react with assay.

Total bilirubin (TBIL) measures both BU and BC. Total and direct bilirubin levels can be measured from the blood, but indirect bilirubin is calculated from the total and direct bilirubin.

Hope this info will help you.

Good luck!

Ojala y te pueda ayudar pero creo que el calculo de la bilirrubina indirecta se obtiene a partir de la bilirrubina total y la directa

Karen A. Darbinyan thanks for your answer and clarification

So, if I want to measure the unconjugated bilirubin in serum, I have to measure total bilirubin and subtract the conjugated bilirubin?

unconjugated bilirubin in serum= total bilirubin in serum - conjugated bilirubin in serum

I also read that in serum the level of the conjugated bilirubin should be theoretically very low, because it is formed in the liver. So, in this case, measuring the tot bilirubin correspond with the unconjugated bilirubin? I am asking that because I don't have a lot of serum samples to use....If I can do only one measurement, that would help!

Hello yes unconjugated bilirubin in serum= total bilirubin in serum - conjugated bilirubin in serum.

As in the answers above, the indirect bilirubin can be calculated from total and direct. However, I think that IB may not be suitable to detect a low level of hemolysis since this calculation can be affected by analytical errors even small of the two measurements (TB + DB). So, If you need a qualitative evaluation only (yes/no hemolysis), you can use the measurement of haptoglobin (HPG, decreased after hemolysis ) and/or LDH (increased). HPG is more specific and sensitive; LDH is sensitive but less specific.

If you need to evaluate hemolysis quantitatively the RBC counting before and after hemolysis can be an excellent indicator (i.e. RBC before - RBC after = number of hemolysed RBC).

The confusing terminologies are responsible for your question. Remember like this:

1. Bilirubin newly formed and delivered in plasma = Bound to proteins (mainly albumin) = Water insoluble = Does not appear in urine = UNCONJUGATED; because conjugation with glucuronic acid has yet not taken in liver = This DOES NOT react with diazotized sulfanilic acid test directly without adding facilitating reagent like caffeine, hence laboratory people call it as INDIRECT.

2. Next it is processed in liver; splits from albumin and CONJUGATED (combined) with two molecules of Glucuronic acid, making it water soluble and excreted in bile. As this fraction is water soluble, gives the chemical reaction with diazotized sulfanilic acid directly without caffeine; Hence described as DIRECT bilirubin. This fraction can appear in urine.

3. When you measure bilirubin in presence of caffeine, it measures TOTAL bilirubin.

4. So you test bilirubin twice; once without caffeine (Direct or Unconjugated), and second time with caffeine reagent (TOTAL)

5. Indirect (Unconjugated) bilirubin is calculated as the difference of these two.

I hope I have not increased the confusion.

Ramesh Z. Patel

Thanks for the specification. I got the terminology. My question is:

- UNCONJUGATED /INDIRECT bilirubin needs caffeine to be detected.

- CONJUGATED /DIRECT bilirubin DOES NOT need caffeine to be detected. Plus, it is formed in the liver.

So, if I measure bilirubin in serum, WITH addition of caffeine I will find UNCONJUGATED /INDIRECT bilirubin that needs caffeine to be detected? Can I make this conclusion? Also because the CONJUGATED /DIRECT bilirubin should not be found in the serum but in the liver. Am I right?

No, Anna. You can obtain the indirect bilirubin only as a difference between total and direct bilirubin. In other words, there are no commercial kits available to measure unconjugated bilirubin directly but kits that contain some agents that facilitate the diazo reaction (such as caffeine or alcohol) for total bilirubin measuring and kits without caffeine or alcohol for direct (conjugated) bilirubin. However, I repeat that the measurement of indirect bilirubin, in whatever way is obtained, is not usefull for the quantification of hemolysis. Beside, the same indirect bilirubin may give indirect and rough information only on the presence of hemolysis and only on condition that this is relevant

Most of the bilirubin conjugated in liver is excreted in bile, but a very small fraction (not more than 0.2 mg/100ml) remains in plasma. As such in health, most of the bilirubin formed is conjugated by liver, barring a very small amount (not more than 0.8 mg/100ml) of unconjugated bilirubin, which circulates in plasma bound with albumin in healthy humans. In case of mild hemolysis (extra-vascular), bilirubin does not increase, because liver has reserve capacity to process some extra bilirubin. This

In such cases, serum LDH and urinary urobilirubinogen increase. But remember when serum bilirubin is measured using caffeine reagent not only INDIRECT but also DIRECT bilirubin react with diazotized sulfanilic acid and give pink colour of AZO-BILIRUBIN. At acidic pH it is red, but changes its colour to blue at alkaline pH behaving like pH indicator, and is measured at around 600 nm. In the Jendrassik-Grof method, this is achieved by adding alkaline tartarate reagent.

In mild intra-vascular hemolysis, serum HAPTOGLOBIN level falls and may become zero.

Ramesh Z. Patel Antonio La Gioia

Thanks a lot for your patience and clarification! Which kit wwould you suggest me for the measurement of serum Hapatoglobin?

Thanks again!

Dear Anna

Unfortunately, your analytical problem is more complicated and not easy to resolve. I will try to list the possible solutions with the respective advantages and criticalities.

I hope I didn't complicate your problem. The critical point is the withdrawal mode: If the mouse is sacrificed, the sample collection is easy and without traumatisms that can cause mechanical hemolysis. So you can use either method 3 or the 4 or both.

Antonio La Gioia

Thank you so much for the clarification. Yesterday, I was googling haptoglobin kit and I found this one: https://www.abcam.com/mouse-haptoglobin-elisa-kit-ab157714.html

Do you think it can work? seems good...

Thanks!

Wonderful! I didn't think there were any mouse aptoglobin kits. Good luck with your work. I would like to have information on future results. Thanks for the interesting discussion

Antonio La Gioia

Sure! I will contact you later!

Another question came to my mind. If the total bilirubin is low, can it be already a sign of hemolytic anemia? Is that sufficient?

I am asking that because I don't have a lot of serum. So, I would rather do one measurement than two.

Thanks!

Hello, Before thinking about hemolytic anemia low bilirubin levels can be associated with coronary artery disease, inflammation and arteriosclerosis. Thus, decreased bilirubin levels also associated with angina, which is caused by coronary artery disease and arteriosclerosis as well as inflammation.

Hope this info will help you.

Good luck!

unconjugated bilirubin in serum= total bilirubin in serum - conjugated bilirubin in serum

Increased unconjugated bilirubin production can result from the following:

• Hemolysis
• Dyserythropoiesis
• Hematoma

For all of the already cited diseases, there are not pathophysiological reasons to affirm that these are associated with low levels of bilirubin. The "normal range" of Bil is between 0.2 and 1.0 mg/dL, and it is related to the physiological turnover of the RBCs. Every Increasing of Bil accompanies every increasing of the RBCs turnover. A decreasing of B level cannot exist. So Anna, the kit for murine haptoglobin is a good solution that must convince you of the usefulness of the Biluribine assay for evaluating the hemolysis level in serum.

Antonio La Gioia

Sorry, i meant HIGH bilirubin.

Anyhow, I am more than convinced for haptoglobin test....but for some reasons....I can not do it....

Dear Anna, The use and quality of results that we get in a search depend on the purposes of the research itself and can take on different valence depending on the context. I do not know what the purposes of your search are and what you expect from the results. I do not know how you cause in mice hemolysis in vivo; I do not know if you need to know if the hemolysis happened or if you have to quantify it. Therefore, from a general point of view, I repeat that the dosage of bilirubin is not suitable for the evaluation of hemolysis because even if it increases, it can be correlated with the hemolysis only in a likely way.

Further, you have to have normal range in the animal species. Next how do you going to produce hemolysis ? Is it going to be intravascular or extravascular ? Because bilirubin increase occurs only when the increase in extravascular exceeds the reserve conjugation capacity of liver. Serum haptoglobin is a sensitive parameter for intravascular hemolysys; and then also the data of normal range in the animal by that particular kit is required before you start the project. Hope I have not further confused you.