Editing Bilirubin (section)

== Blood tests ==
Bilirubin is degraded by light. Blood collection tubes containing blood or (especially) serum to be used in bilirubin assays should be protected from illumination.<ref name="Rehak_2007">{{cite journal |last1=Rehak |first1=Nadja N. |last2=Cecco |first2=Stacey A. |last3=Hortin |first3= Glen L. |date=January 2008 |title=Photolysis of bilirubin in serum specimens exposed to room lighting |journal=Clinica Chimica Acta |volume=387 |issue=1–2 |pages=181–183 |doi=10.1016/j.cca.2007.09.019 |pmid=17967443 |pmc=2131702 }}</ref> For adults, blood is typically collected by needle from a vein in the arm.<ref name="Mayo Clinic 2022 c113">{{cite web | title=Bilirubin test: What you can expect | website=Mayo Clinic | date=October 8, 2022 | url=https://www.mayoclinic.org/tests-procedures/bilirubin/about/pac-20393041 | access-date=March 24, 2024}}</ref> In newborns, blood is often collected from a heel stick, a technique that uses a small, sharp blade to cut the skin on the infant's heel and collect a few drops of blood into a small tube. Non-invasive technology is available in some health care facilities that will measure bilirubin by using a bilirubinometer which shines light onto the skin and calculates the amount of bilirubin by analysing how the light is absorbed or reflected.<ref name="nhs.uk 2017 c967">{{cite web | title=Newborn jaundice: Bilirubin test | website=[[National Health Service]] UK | date=September 15, 2017 | url=https://www.nhs.uk/conditions/jaundice-newborn/diagnosis/#:~:text=Bilirubin%20test&text=a%20blood%20test%20of%20a,the%20serum%20is%20then%20measured) | access-date=March 24, 2024}}</ref> This device is also known as a transcutaneous bilirubin meter.<ref name="Lucanova_2021">{{cite journal |last1= Lucanova |first1= Lucia Casnocha |last2=Zibolenova |first2=Jana |last3=Matasova |first3= Katarina |last4=Docekalova |first4=Lenka |last5=Zibolen |first5=Mirko |date=2021-01-01 |title=Accuracy of enhanced transcutaneous bilirubinometry according to various measurement sites |journal=Turkish Archives of Pediatrics |volume=56 |issue=1 |pages=15–21 |doi=10.14744/TurkPediatriArs.2020.54514 |pmid= 34013224 |pmc=8114612 }}</ref>

Bilirubin (in blood) is found in two forms:

{| class="wikitable sortable"
! Abb. || Name(s)  || Water-soluble || Reaction
 |-
 | "BC" ||"Conjugated bilirubin"|| Yes (bound to glucuronic acid) || Reacts quickly when dyes (diazo reagent) are added to the blood specimen to produce [[azobilirubin]] "Direct bilirubin"
 |-
 | "BU" ||"Unconjugated bilirubin"|| No || Reacts more slowly, still produces azobilirubin, Ethanol makes all bilirubin react promptly, then: indirect bilirubin = total bilirubin – direct bilirubin
|}
Note: 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 reagents; direct bilirubin is often made up largely of conjugated bilirubin, but some unconjugated bilirubin (up to 25%) can still be part of the "direct" bilirubin fraction. Likewise, not all conjugated bilirubin is readily available in solution for reaction or detection (for example, if it is hydrogen bonding with itself) and therefore would not be included in the direct bilirubin fraction.{{citation needed|date=November 2021}}

Total bilirubin (TBIL) measures both BU and BC. Total bilirubin assays work by using surfactants and accelerators (like caffeine) to bring all of the different bilirubin forms into solution where they can react with assay reagents. Total and direct bilirubin levels can be measured from the blood, but indirect bilirubin is calculated from the total and direct bilirubin.{{cn|date=July 2024}}

Indirect bilirubin is fat-soluble and direct bilirubin is water-soluble.<ref>{{cite web |title=Bilirubin: The Test {{!}} Bilirubin Test: Total bilirubin; TBIL; Neonatal bilirubin; Direct bilirubin; Conjugated bilirubin; Indirect bilirubin; Unconjugated bilirubin {{!}} Lab Tests Online |url=https://labtestsonline.org/understanding/analytes/bilirubin/tab/test/ |access-date=2017-06-14 |website=[[labtestsonline.org]] |language=en-US}}</ref>

=== Total bilirubin ===
Total bilirubin = direct bilirubin + indirect bilirubin<ref name="Tietze 2012 pp. 86–122">{{cite book |url=https://archive.org/details/clinicalskillsfo0000tiet |title=Clinical Skills for Pharmacists |vauthors=Tietze KJ |publisher=Elsevier |year=2012 |isbn=978-0-323-07738-5 |pages=[https://archive.org/details/clinicalskillsfo0000tiet/page/86 86]–122 |chapter=Review of Laboratory and Diagnostic Tests |doi=10.1016/b978-0-323-07738-5.10005-5 |url-access=registration}}</ref>

Elevation of both [[alanine aminotransferase]] (ALT) and bilirubin is more indicative of serious liver injury than is elevation in ALT alone, as postulated in [[Hy's law]] that elucidates the relation between the lab test results and [[drug-induced liver injury]]<ref name="Gwaltney-Brant 2016 pp. 87–99">{{cite book |title=Nutraceuticals |vauthors=Gwaltney-Brant SM |publisher=Elsevier |year=2016 |isbn=978-0-12-802147-7 |pages=87–99 |chapter=Nutraceuticals in Hepatic Diseases |doi=10.1016/b978-0-12-802147-7.00007-3 |s2cid=78381597}}</ref>

=== Indirect (unconjugated) ===

The measurement of unconjugated bilirubin (UCB) is underestimated by measurement of indirect bilirubin, as unconjugated bilirubin (without/yet glucuronidation) reacts with diazosulfanilic acid to create [[azobilirubin]] which is measured as direct bilirubin.<ref name="Medscape Reference 2019">{{cite web |date=2019-03-04 |title=Unconjugated Hyperbilirubinemia: Practice Essentials, Background, Pathophysiology |url=https://emedicine.medscape.com/article/178841-overview#a2 |access-date=2019-05-06 |website=Medscape Reference}}</ref><ref name="Medscape Reference 2019 II">{{cite web |date=2019-02-01 |title=Bilirubin: Reference Range, Interpretation, Collection and Panels |url=https://emedicine.medscape.com/article/2074068-overview |access-date=2019-05-06 |website=Medscape Reference}}</ref>

===Direct===

Direct bilirubin = Conjugated bilirubin + delta bilirubin<ref name="Tietze 2012 pp. 86–122" />

==== Conjugated ====
In the liver, bilirubin is conjugated with [[glucuronic acid]] by the enzyme [[glucuronyltransferase]], first to [[bilirubin glucuronide]] and then to [[bilirubin diglucuronide]], making it soluble in water: the conjugated version is the main form of bilirubin present in the "direct" bilirubin fraction. Much of it goes into the bile and thus out into the small intestine. Though most [[bile acid]] is reabsorbed in the [[terminal ileum]] to participate in [[enterohepatic circulation]], conjugated bilirubin is not absorbed and instead passes into the [[Large intestine|colon]].<ref name="handbook">{{cite book |title=Oxford American Handbook of Gastroenterology and Hepatology |vauthors=Cheifetz AS |publisher=Oxford University Press, USA |year=2010 |isbn=978-0199830121 |location=Oxford |page=165}}</ref>

There, colonic bacteria deconjugate and metabolize the bilirubin into colorless [[urobilinogen]], which can be oxidized to form [[urobilin]] and [[stercobilin]]. Urobilin is excreted by the kidneys to give urine its yellow color and stercobilin is excreted in the feces giving stool its characteristic brown color. A trace (~1%) of the urobilinogen is reabsorbed into the [[enterohepatic circulation]] to be re-excreted in the bile.<ref>{{cite book |last=Kuntz |first=Erwin |title=Hepatology: Textbook and Atlas |publisher=Springer |year=2008 |isbn=978-3-540-76838-8 |location=Germany |page=38}}</ref>

Conjugated bilirubin's half-life is shorter than delta bilirubin.<ref name="Sullivan Gourley 2011 pp. 176–186.e3">{{cite book |title=Pediatric Gastrointestinal and Liver Disease |vauthors=Sullivan KM, Gourley GR |publisher=Elsevier |year=2011 |isbn=978-1-4377-0774-8 |pages=176–186.e3 |chapter=Jaundice |doi=10.1016/b978-1-4377-0774-8.10017-x}}</ref>

====Delta bilirubin====

Although the terms direct and indirect bilirubin are sometimes used interchangeably with conjugated and unconjugated bilirubin, the direct fraction actually includes both conjugated bilirubin and delta bilirubin.<ref>{{Citation |last=Sullivan |first=Kara M. |title=17 - Jaundice |date=2011-01-01 |work=Pediatric Gastrointestinal and Liver Disease (Fourth Edition) |pages=176–186.e3 |editor-last=Wyllie |editor-first=Robert |url=https://linkinghub.elsevier.com/retrieve/pii/B978143770774810017X |access-date=2025-02-14 |place=Saint Louis |publisher=W.B. Saunders |doi=10.1016/b978-1-4377-0774-8.10017-x |isbn=978-1-4377-0774-8 |last2=Gourley |first2=Glenn R. |editor2-last=Hyams |editor2-first=Jeffrey S.|url-access=subscription }}</ref>

Delta bilirubin is albumin-bound conjugated bilirubin.<ref name="Tietze 2012 pp. 86–122" /> In the other words, delta bilirubin is the kind of bilirubin covalently bound to [[albumin]], which appears in the serum when hepatic excretion of conjugated bilirubin is impaired in patients with [[hepatobiliary disease]].<ref>{{cite book |title=Nelson Textbook of Pediatrics |vauthors=Moyer KD, Balistreri WF |publisher=Saunders |year=2011 |isbn=978-1-4377-0755-7 |veditors=Kliegman RM, Stanton BF, St Geme JW, Schor NF, Behrman RE |page=1405 |chapter=Liver Disease Associated with Systemic Disorders |chapter-url=http://www.mdconsult.com/books/page.do?eid=4-u1.0-B978-1-4377-0755-7..00352-3&isbn=978-1-4377-0755-7&type=bookPage&from=content&uniqId=433360670-2}}</ref> Furthermore, direct bilirubin tends to overestimate conjugated bilirubin levels due to unconjugated bilirubin that has reacted with diazosulfanilic acid, leading to increased azobilirubin levels (and increased direct bilirubin).{{cn|date=July 2024}}

δ bilirubin = total bilirubin – (unconjugated bilirubin + conjugated bilirubin)<ref name="Tietze 2012 pp. 86–122" />

=====Half-life=====

The half-life of delta bilirubin is equivalent to that of [[human serum albumin|albumin]] since the former is bound to the latter, yields 2–3 weeks.<ref name="Kalakonda John 2019 p.">{{cite book |title=StatPearls |vauthors=Kalakonda A, John S |publisher=StatPearls Publishing |year=2019 |location=Treasure Island (FL) |chapter=Physiology, Bilirubin article-18281 |pmid=29261920 |quote=This fraction of conjugated bilirubin gets covalently bound to albumin, and is called delta bilirubin or delta fraction or biliprotein. As the delta bilirubin is bound to albumin, its clearance from serum takes about 12–14 days (equivalent to the half-life of albumin) in contrast to the usual 2 to 4 hours (half-life of bilirubin). |access-date=2019-12-22 |chapter-url=http://www.ncbi.nlm.nih.gov/books/NBK470290/}}</ref><ref name="Medscape Reference 2019" />

A free-of-bound bilirubin has a half-life of 2 to 4 hours.<ref name="Kalakonda John 2019 p." />

{{further|Bilirubin glucuronide}}

=== Measurement methods ===
Originally, the [[Van den Bergh reaction]] was used for a qualitative estimate of bilirubin.{{cn|date=July 2024}}

This test is performed routinely in most [[Medical laboratory|medical laboratories]] and can be measured by a variety of methods.<ref>{{cite journal |vauthors=Watson D, Rogers JA |date=May 1961 |title=A study of six representative methods of plasma bilirubin analysis |journal=[[Journal of Clinical Pathology]] |volume=14 |issue=3 |pages=271–8 |doi=10.1136/jcp.14.3.271 |pmc=480210 |pmid=13783422}}</ref>

Total bilirubin is now often measured by the 2,5-dichlorophenyldiazonium (DPD) method, and direct bilirubin is often measured by the method of Jendrassik and Grof.<ref>{{cite journal |vauthors=Rolinski B, Küster H, Ugele B, Gruber R, Horn K |date=October 2001 |title=Total bilirubin measurement by photometry on a blood gas analyzer: potential for use in neonatal testing at the point of care |url=http://www.clinchem.org/cgi/pmidlookup?view=long&pmid=11568098 |journal=[[Clinical Chemistry (journal)|Clinical Chemistry]] |volume=47 |issue=10 |pages=1845–7 |doi=10.1093/clinchem/47.10.1845 |pmid=11568098 |doi-access=free}}</ref>