Phototherapy is the standard treatment for neonatal hyperbilirubinemia. During phototherapy, the highly lipophilic bilirubin is converted into more hydrophilic photoisomers, which can be more easily excreted from the body. This process typically lowers bilirubin levels to non-harmful concentrations. However, despite decades of research into the formation and role of bilirubin photoisomers, methodological limitations and the compound's complex biochemistry have hindered comprehensive understanding. This review provides an updated overview of current knowledge on bilirubin photoisomers, including their basic chemistry, analytical quantification, clinical relevance, and future research directions. Improved insight into the mechanism of photoisomer formation and kinetics may inform optimization of phototherapy parameters, including light intensity and wavelength, and offer additional indicators of treatment efficacy beyond total bilirubin concentration. Advances in sensitive and standardized mass spectrometry techniques now enable more accurate measurement of different bilirubin isomers and serve as a first step towards a deeper insight into the clinical relevance of photoisomers.

Department of Clinical Chemistry Karolinska University Hospital 171 64 Stockholm Sweden

Department of Laboratory Medicine Karolinska Institutet 171 77 Stockholm Sweden

Department of Laboratory Medicine Radboud University medical center 6525 GA Nijmegen The Netherlands

Division of Neonatology Department of Pediatrics Beatrix Children's Hospital University Medical Centre Groningen 9713 GZ Groningen The Netherlands

Institute of Medical Biochemistry and Laboratory Diagnostics and 4th Department of Internal Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague 121 08 Prague Czech Republic

Murdoch Children's Research Institute Parkville VIC 3052 Australia

School of Health Sciences Swinburne University of Technology Melbourne VIC 3122 Australia

Zobrazit více v PubMed

Cremer R.J., Perryman P.W., Richards D.H. Influence of light on the hyperbilirubinaemia of infants. Lancet. 1958;271:1094–1097. doi: 10.1016/S0140-6736(58)91849-X. PubMed DOI

Ferreira H.C., Cardim W.H., Mellone O. Phototherapy. A new therapeutic method in hyperbilirubinemia of the newborn. J. Pediatr. 1960;25:347–391. PubMed

Lucey J., Ferreiro M., Hewitt J. Prevention of hyperbilirubinemia of prematurity by phototherapy. Pediatrics. 1968;41:1047–1054. doi: 10.1542/peds.41.6.1047. PubMed DOI

Scheidt P.C., Bryla D.A., Nelson K.B., Hirtz D.G., Hoffman H.J. Phototherapy for Neonatal Hyperbilirubinemia—6-Year Follow-up of the National-Institute-of-Child-Health-and-Human-Development Clinical-Trial. Pediatrics. 1990;85:455–463. PubMed

Bergman D.A., Cooley J.R., Coombs J.B., Goldberg M.J., Homer C.J., Nazarian L.F., Riemenschneider T.A., Roberts K.B., Shea D.W., Tonniges T.F., et al. Practice Parameter—Management of Hyperbilirubinemia in the Healthy Term Newborn. Pediatrics. 1994;94:558–565. PubMed

Morris B.H., Oh W., Tyson J.E., Stevenson D.K., Phelps D.L., O’Shea T.M., McDavid G.E., Perritt R.L., Van Meurs K.P., Vohr B.R., et al. Aggressive vs. conservative phototherapy for infants with extremely low birth weight. N. Engl. J. Med. 2008;359:1885–1896. doi: 10.1056/NEJMoa0803024. PubMed DOI PMC

Jasprová J., Dal Ben M., Hurny D., Hwang S., Zízalová K., Kotek J., Wong R.J., Stevenson D.K., Gazzin S., Tiribelli C., et al. Neuro-inflammatory effects of photodegradative products of bilirubin. Sci. Rep. 2018;8:7444. doi: 10.1038/s41598-018-25684-2. PubMed DOI PMC

Mujawar T., Sevelda P., Madea D., Klan P., Svenda J. A Platform for the Synthesis of Oxidation Products of Bilirubin. J. Am. Chem. Soc. 2024;146:1603–1611. doi: 10.1021/jacs.3c11778. PubMed DOI PMC

Ennever J.F., Costarino A.T., Polin R.A., Speck W.T. Rapid Clearance of a Structural Isomer of Bilirubin during Phototherapy. J. Clin. Investig. 1987;79:1674–1678. doi: 10.1172/JCI113006. PubMed DOI PMC

McDonagh A.F. Bilirubin photo-isomers: Regiospecific acyl glucuronidation in vivo. Monatshefte Fur. Chem. 2014;145:465–482. doi: 10.1007/s00706-013-1076-6. DOI

Vreman H.J., Kourula S., Jasprová J., Ludvíková L., Klán P., Muchová L., Vítek L., Cline B.K., Wong R.J., Stevenson D.K. The effect of light wavelength on in vitro bilirubin photodegradation and photoisomer production. Pediatr. Res. 2019;85:865–873. doi: 10.1038/s41390-019-0310-2. Correction in Pediatr. Res. 2019, 85, 905. PubMed DOI

Uchida Y., Takahashi Y., Kurata C., Morimoto Y., Ohtani E., Tosaki A., Kumagai A., Greimel P., Nishikubo T., Miyawaki A. Urinary lumirubin excretion in jaundiced preterm neonates during phototherapy with blue light-emitting diode vs. green fluorescent lamp. Sci. Rep. 2023;13:18359. doi: 10.1038/s41598-023-45147-7. PubMed DOI PMC

Takahashi M., Sugiyama K., Shumiya S., Nagase S. Penetration of Bilirubin into the Brain in Albumin-Deficient and Jaundiced Rats (Ajr) and Nagase Analbuminemic Rats (Nar) J. Biochem. 1984;96:1705–1712. doi: 10.1093/oxfordjournals.jbchem.a135003. PubMed DOI

Wennberg R.P. The blood-brain barrier and bilirubin encephalopathy. Cell. Mol. Neurobiol. 2000;20:97–109. doi: 10.1023/A:1006900111744. PubMed DOI PMC

Lightner D.A., Reisinger M., Landen G.L. On the Structure of Albumin-Bound Bilirubin—Selective Binding of Intramolecularly Hydrogen-Bonded Conformational Enantiomers. J. Biol. Chem. 1986;261:6034–6038. doi: 10.1016/S0021-9258(17)38489-2. PubMed DOI

Moss G.P. Basic terminology of stereochemistry. Pure Appl. Chem. 1996;68:2193–2222. doi: 10.1351/pac199668122193. DOI

McDonagh A.F., Assisi F. Ready Isomerization of Bilirubin-IX-Alpha in Aqueous-Solution. Biochem. J. 1972;129:797–800. doi: 10.1042/bj1290797. PubMed DOI PMC

McDonagh A.F. Ex uno plures: The concealed complexity of bilirubin species in neonatal blood samples. Pediatrics. 2006;118:1185–1187. doi: 10.1542/peds.2006-0594. PubMed DOI

Ullrich D., Fevery J., Sieg A., Tischler T., Bircher J. The Influence of Gestational-Age on Bilirubin Conjugation in Newborns. Eur. J. Clin. Investig. 1991;21:83–89. doi: 10.1111/j.1365-2362.1991.tb01363.x. PubMed DOI

Ennever J.F., Knox I., Denne S.C., Speck W.T. Phototherapy for Neonatal Jaundice—Invivo Clearance of Bilirubin Photoproducts. Pediatr. Res. 1985;19:205–208. doi: 10.1203/00006450-198502000-00012. PubMed DOI

Itoh S., Onishi S. Kinetic study of the photochemical changes of (ZZ)-bilirubin IX alpha bound to human serum albumin. Demonstration of (EZ)-bilirubin IX alpha as an intermediate in photochemical changes from (ZZ)-bilirubin IX alpha to (EZ)-cyclobilirubin IX alpha. Biochem. J. 1985;226:251–258. doi: 10.1042/bj2260251. PubMed DOI PMC

Onishi S., Kawade N., Itoh S., Isobe K., Sugiyama S. High-Pressure Liquid-Chromatographic Analysis of Anaerobic Photoproducts of Bilirubin-Ix-Alpha Invitro and Its Comparison with Photoproducts Invivo. Biochem. J. 1980;190:527–532. doi: 10.1042/bj1900527. PubMed DOI PMC

Bhutani V.K., Wong R.J., Turkewitz D., Rauch D.A., Mowitz M.E., Barfield W.D., Eichenwald E., Ambalavanan N., Guillory C., Hudak M. Phototherapy to prevent severe neonatal hyperbilirubinemia in the newborn infant 35 or more weeks of gestation: Technical report. Pediatrics. 2024;154:e2024068026. doi: 10.1542/peds.2024-068026. PubMed DOI

Ebbesen F., Hansen T.W., Maisels M.J. Update on phototherapy in jaundiced neonates. Curr. Pediatr. Rev. 2017;13:176–180. doi: 10.2174/1573396313666170718150056. PubMed DOI

Ebbesen F., Madsen P.H., Rodrigo-Domingo M., Donneborg M.L. Bilirubin isomers during LED phototherapy of hyperbilirubinemic neonates, blue-green (∼478 nm) vs blue. Pediatr. Res. 2024;97:1623–1628. doi: 10.1038/s41390-024-03493-w. PubMed DOI

Cruz A.B., de Brito L.G., Leal P.V.B., Ramos W.T.D., Pereira D.H. Intramolecular hydrogen bonds interactions in the isomers of the bilirubin molecule: DFT and QTAIM analysis. J. Mol. Model. 2023;29:318. doi: 10.1007/s00894-023-05720-3. PubMed DOI

Troup G.J., Agati G., Fusi F., Pratesi R. Photophysics of the variable quantum yield of asymmetric bilirubin. Aust. J. Phys. 1996;49:673–681. doi: 10.1071/PH960673. DOI

Ebbesen F., Madsen P.H., Vandborg P.K., Jakobsen L.H., Trydal T., Vreman H.J. Bilirubin isomer distribution in jaundiced neonates during phototherapy with LED light centered at 497 nm (turquoise) vs. 459 nm (blue) Pediatr. Res. 2016;80:511–515. doi: 10.1038/pr.2016.115. PubMed DOI

Lamola A.A. Optical Properties and Structure of Tetrapyrroles. De Gruyter Brill; Berlin, Germany: 1985. Effects of environment on photophysical processes of bilirubin; pp. 311–326.

Lamola A.A., Flores J., Doleiden F.H. Quantum Yield and Equilibrium Position of the Configurational Photo-Isomerization of Bilirubin Bound to Human-Serum Albumin. Photochem. Photobiol. 1982;35:649–654. doi: 10.1111/j.1751-1097.1982.tb02624.x. PubMed DOI

Dixon J.M., Taniguchi M., Lindsey J.S. PhotochemCAD 2: A Refined Program with Accompanying Spectral Databases for Photochemical Calculations. Photochem. Photobiol. 2005;81:212–213. doi: 10.1111/j.1751-1097.2005.tb01544.x. PubMed DOI

Du H., Fuh R.C.A., Li J.Z., Corkan L.A., Lindsey J.S. PhotochemCAD: A computer-aided design and research tool in photochemistry. Photochem. Photobiol. 1998;68:141–142. doi: 10.1111/j.1751-1097.1998.tb02480.x. DOI

Lee K.S., Gartner L.M. Spectrophotometric Characteristics of Bilirubin. Pediatr. Res. 1976;10:782–788. doi: 10.1203/00006450-197609000-00004. PubMed DOI

Weisiger R.A., Ostrow J.D., Koehler R.K., Webster C.C., Mukerjee P., Pascolo L., Tiribelli C. Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: Results of a novel ultrafiltration method. J. Biol. Chem. 2001;276:29953–29960. doi: 10.1074/jbc.M104628200. PubMed DOI

Zunszain P.A., Ghuman J., McDonagh A.F., Curry S. Crystallographic analysis of human serum albumin complexed with 4Z,15E-bilirubin-IXalpha. J. Mol. Biol. 2008;381:394–406. doi: 10.1016/j.jmb.2008.06.016. PubMed DOI PMC

Nii K., Okada H., Itoh S., Kusaka T. Characteristics of bilirubin photochemical changes under green light-emitting diodes in humans compared with animal species. Sci. Rep. 2021;11:6391. doi: 10.1038/s41598-021-85632-5. PubMed DOI PMC

Cuperus F.J., Schreuder A.B., van Imhoff D.E., Vitek L., Vanikova J., Konickova R., Ahlfors C.E., Hulzebos C.V., Verkade H.J. Beyond plasma bilirubin: The effects of phototherapy and albumin on brain bilirubin levels in Gunn rats. J. Hepatol. 2013;58:134–140. doi: 10.1016/j.jhep.2012.08.011. PubMed DOI

Nagase S., Shimamune K., Shumiya S. Albumin-deficient rat mutant. Science. 1979;205:590–591. doi: 10.1126/science.451621. PubMed DOI

Vodret S., Bortolussi G., Schreuder A.B., Jasprova J., Vitek L., Verkade H.J., Muro A.F. Albumin administration prevents neurological damage and death in a mouse model of severe neonatal hyperbilirubinemia. Sci. Rep. 2015;5:16203. doi: 10.1038/srep16203. PubMed DOI PMC

Mitra S., Samanta M., Sarkar M., De A.K., Chatterjee S. Pre-exchange 5% albumin infusion in low birth weight neonates with intensive phototherapy failure--a randomized controlled trial. J. Trop. Pediatr. 2011;57:217–221. doi: 10.1093/tropej/fmq083. PubMed DOI

Shahian M., Moslehi M.A. Effect of albumin administration prior to exchange transfusion in term neonates with hyperbilirubinemia--a randomized controlled trial. Indian Pediatr. 2010;47:241–244. doi: 10.1007/s13312-010-0046-x. PubMed DOI

Dash N., Kumar P., Sundaram V., Attri S.V. Pre exchange Albumin Administration in Neonates with Hyperbilirubinemia: A Randomized Controlled Trial. Indian Pediatr. 2015;52:763–767. doi: 10.1007/s13312-015-0713-z. PubMed DOI

Magai D.N., Mwaniki M., Abubakar A., Mohammed S., Gordon A.L., Kalu R., Mwangi P., Koot H.M., Newton C.R. A randomized control trial of phototherapy and 20% albumin versus phototherapy and saline in Kilifi, Kenya. BMC Res. Notes. 2019;12:617. doi: 10.1186/s13104-019-4632-2. PubMed DOI PMC

Govaert P., Lequin M., Swarte R., Robben S., De Coo R., Weisglas-Kuperus N., De Rijke Y., Sinaasappel M., Barkovich J. Changes in globus pallidus with (Pre) term kernicterus. Pediatrics. 2003;112:1256–1263. doi: 10.1542/peds.112.6.1256. PubMed DOI

Kemper A.R., Newman T.B., Slaughter J.L., Maisels M.J., Watchko J.F., Downs S.M., Grout R.W., Bundy D.G., Stark A.R., Bogen D.L., et al. Clinical Practice Guideline Revision: Management of Hyperbilirubinemia in the Newborn Infant 35 or More Weeks of Gestation. Pediatrics. 2022;150:e2022058859. doi: 10.1542/peds.2022-058859. PubMed DOI

Hansen T.W.R., Maisels M.J., Ebbesen F., Vreman H.J., Stevenson D.K., Wong R.J., Bhutani V.K. Sixty years of phototherapy for neonatal jaundice–from serendipitous observation to standardized treatment and rescue for millions. J. Perinatol. 2020;40:180–193. doi: 10.1038/s41372-019-0439-1. PubMed DOI

Hansen T.W.R. Biology of bilirubin photoisomers. Clin. Perinatol. 2016;43:277–290. doi: 10.1016/j.clp.2016.01.011. PubMed DOI

Hansen T.W.R. Seminars in Perinatology. Volume 34. Elsevier; Amsterdam, The Netherlands: 2010. Phototherapy for neonatal jaundice—Therapeutic effects on more than one level? pp. 231–234. PubMed

Capková N., Pospísilová V., Fedorová V., Raska J., Pospísilová K., Dal Ben M., Dvorák A., Viktorová J., Bohaciaková D., Vítek L. The Effects of Bilirubin and Lumirubin on the Differentiation of Human Pluripotent Cell-Derived Neural Stem Cells. Antioxidants. 2021;10:1532. doi: 10.3390/antiox10101532. PubMed DOI PMC

Doumas B.T., Wu T.W., Jendrzejczak B. Delta-Bilirubin—Absorption-Spectra, Molar Absorptivity, and Reactivity in the Diazo Reaction. Clin. Chem. 1987;33:769–774. doi: 10.1093/clinchem/33.6.769. PubMed DOI

Kiuchi S., Ihara H., Osawa S., Ishibashi M., Kinpara K., Ohtake K., Ida T., Miura Y., Fujimura Y., Ueda S., et al. A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin. Ann. Clin. Biochem. 2021;58:563–571. doi: 10.1177/00045632211026699. PubMed DOI

Kawamoto S., Koyano K., Ozaki M., Arai T., Iwase T., Okada H., Itoh S., Murao K., Kusaka T. Effects of bilirubin configurational photoisomers on the measurement of direct bilirubin by the vanadate oxidation method. Ann. Clin. Biochem. 2021;58:311–317. doi: 10.1177/0004563221999068. PubMed DOI

Okada H., Itoh S., Kawamoto S., Ozaki M., Kusaka T. Reactivity of bilirubin photoisomers on the measurement of direct bilirubin using vanadic acid method. Ann. Clin. Biochem. 2018;55:296–298. doi: 10.1177/0004563217709844. PubMed DOI

Itoh S., Kusaka T., Imai T., Isobe K., Onishi S. Effects of bilirubin and its photoisomers on direct bilirubin measurement using bilirubin oxidase. Ann. Clin. Biochem. 2000;37:452–456. doi: 10.1177/000456320003700404. PubMed DOI

Kawaguchi N., Koyano K., Morita H., Fadly D., Shinabe Y., Noguchi Y., Arioka M., Nakao Y., Ozaki M., Nakamura S., et al. Quantitative effects of bilirubin photoisomers on the measurement of direct bilirubin by the enzymatic bilirubin oxidase method. Ann. Clin. Biochem. doi: 10.1177/00045632251367245. 2025, online ahead of print. PubMed DOI

Okada H., Kawada K., Itoh S., Ozaki M., Kakutani I., Arai T., Koyano K., Yasuda S., Iwase T., Murao K. Effects of bilirubin photoisomers on the measurement of direct bilirubin by the bilirubin oxidase method. Ann. Clin. Biochem. 2018;55:276–280. doi: 10.1177/0004563217716474. PubMed DOI

Itoh S., Isobe K., Onishi S. Accurate and sensitive high-performance liquid chromatographic method for geometrical and structural photoisomers of bilirubin IXα using the relative molar absorptivity values. J. Chromatogr. A. 1999;848:169–177. doi: 10.1016/S0021-9673(99)00469-0. PubMed DOI

Jasprová J., Dvorák A., Vecka M., Lenícek M., Lacina O., Valáskova P., Zapadlo M., Plavka R., Klán P., Vítek L. A novel accurate LC-MS/MS method for quantitative determination of Z-lumirubin. Sci. Rep. 2020;10:4411. doi: 10.1038/s41598-020-61280-z. PubMed DOI PMC

McCarthy J.J., McClintock S.A., Purdy W.C. The Separation of Bilirubin, Photobilirubin and Their Major Isomers by Ion-Pair High-Pressure Liquid-Chromatography. Anal. Lett. Part. B-Clin. Biochem. Anal. 1984;17:1843–1855. doi: 10.1080/00032718408077187. DOI

McDonagh A.F., Palma L.A., Trull F.R., Lightner D.A. Phototherapy for Neonatal Jaundice—Configurational Isomers of Bilirubin. J. Am. Chem. Soc. 1982;104:6865–6867. doi: 10.1021/ja00388a103. DOI

Moosavi-Movahedi Z., Safarian S., Zahedi M., Sadeghi M., Saboury A.A., Chamani J., Bahrami H., Ashraf-Modarres A., Moosavi-Movahedi A.A. Calorimetric and binding dissections of HSA upon interaction with bilirubin. Protein J. 2006;25:193–201. doi: 10.1007/s10930-006-9002-y. PubMed DOI

Thomas M., Hardikar W., Greaves R.F., Tingay D.G., Loh T.P., Ignjatovic V., Newall F., Rajapaksa A.E. Mechanism of bilirubin elimination in urine: Insights and prospects for neonatal jaundice. Clin. Chem. Lab. Med. 2021;59:1025–1033. doi: 10.1515/cclm-2020-1759. PubMed DOI

Uchida Y., Takahashi Y., Morimoto Y., Greimel P., Tosaki A., Kumagai A., Nishikubo T., Miyawaki A. Noninvasive monitoring of bilirubin photoisomer excretion during phototherapy. Sci. Rep. 2022;12:11798. doi: 10.1038/s41598-022-16180-9. PubMed DOI PMC