Porphyrin biosynthesis

• early mitochondrial:
  • D-Aminolevulinic acid
    • δ-Aminolevulinic acid (also dALA, δ-ALA, 5ALA or 5-aminolevulinic acid), an endogenous non-proteinogenic amino acid, is the first compound in the porphyrin synthesis pathway, the pathway that leads to heme^{[Gardener LC, Cox TM (1988). “Biosynthesis of heme in immature erythroid cells”. The Journal of Biological Chemistry. 263: 6676–6682. doi:10.1016/S0021-9258(18)68695-8.]} in mammals, as well as chlorophyll^{[Von Wettstein D, Gough S, Kannangara CG (July 1995). “Chlorophyll Biosynthesis”. The Plant Cell. 7 (7): 1039–1057. doi:10.1105/tpc.7.7.1039. PMC 160907. PMID 12242396.]} in plants. 5ALA is used in photodynamic detection and surgery of cancer.^{[Wagnières, G.., Jichlinski, P., Lange, N., Kucera, P., Van den Bergh, H. (2014). Detection of Bladder Cancer by Fluorescence Cystoscopy: From Bench to Bedside – the Hexvix Story. Handbook of Photomedicine, 411-426.][Eyüpoglu IY, Buchfelder M, Savaskan NE (March 2013). “Surgical resection of malignant gliomas-role in optimizing patient outcome”. Nature Reviews. Neurology. 9 (3): 141–151. doi:10.1038/nrneurol.2012.279. PMID 23358480. S2CID 20352840.][Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ (May 2006). “Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial”. The Lancet. Oncology. 7 (5): 392–401. doi:10.1016/s1470-2045(06)70665-9. PMID 16648043.][Eyüpoglu IY, Hore N, Savaskan NE, Grummich P, Roessler K, Buchfelder M, Ganslandt O (2012). “Improving the extent of malignant glioma resection by dual intraoperative visualization approach”. PLOS ONE. 7 (9): e44885. Bibcode:2012PLoSO…744885E.]} As a precursor of a photosensitizer, 5ALA is also used as an add-on agent for photodynamic therapy.^{[Yew YW, Lai YC, Lim YL, Chong WS, Theng C (June 2016). “Photodynamic Therapy With Topical 5% 5-Aminolevulinic Acid for the Treatment of Truncal Acne in Asian Patients”. Journal of Drugs in Dermatology. 15 (6): 727–732. PMID 27272080.]} In contrast to larger photosensitizer molecules, it is predicted by computer simulations to be able to penetrate tumor cell membranes.^{[Erdtman E (2008). “Modelling the behavior of 5-aminolevulinic acid and its alkyl esters in a lipid bilayer”. Chemical Physics Letters. 463 (1–3): 178. Bibcode:2008CPL…463..178E. doi:10.1016/j.cplett.2008.08.021.]} Photodynamic detection is the use of photosensitive drugs with a light source of the right wavelength for the detection of cancer, using fluorescence of the drug. 5ALA, or derivatives thereof, can be used to visualize bladder cancer by fluorescence imaging.^{[Wagnières, G.., Jichlinski, P., Lange, N., Kucera, P., Van den Bergh, H. (2014). Detection of Bladder Cancer by Fluorescence Cystoscopy: From Bench to Bedside – the Hexvix Story. Handbook of Photomedicine, 411-426.]}
    • Aminolevulinic acid is indicated in adults for visualization of malignant tissue during surgery for malignant glioma (World Health Organization grade III and IV).^{[“Gliolan EPAR”. European Medicines Agency (EMA). 17 September 2018. Retrieved 6 January 2021.]} It is used to visualise tumorous tissue in neurosurgical procedures.^{[Eyüpoglu IY, Buchfelder M, Savaskan NE (March 2013). “Surgical resection of malignant gliomas-role in optimizing patient outcome”. Nature Reviews. Neurology. 9 (3): 141–151. doi:10.1038/nrneurol.2012.279. PMID 23358480. S2CID 20352840.]} Studies since 2006 have shown that the intraoperative use of this guiding method may reduce the tumour residual volume and prolong progression-free survival in people with malignant gliomas.^{[Stummer W, Pichlmeier U, Meinel T, Wiestler OD, Zanella F, Reulen HJ (May 2006). “Fluorescence-guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled multicentre phase III trial”. The Lancet. Oncology. 7 (5): 392–401. doi:10.1016/s1470-2045(06)70665-9. PMID 16648043.][Eyüpoglu IY, Hore N, Savaskan NE, Grummich P, Roessler K, Buchfelder M, Ganslandt O (2012). “Improving the extent of malignant glioma resection by dual intraoperative visualization approach”. PLOS ONE. 7 (9): e44885. Bibcode:2012PLoSO…744885E.]} The US FDA approved aminolevulinic acid hydrochloride (ALA HCL) for this use in 2017.^{[FDA Approves Fluorescing Agent for Glioma Surgery.June 2017]} Side effects may include liver damage and nerve problems.^{[Qumseya BJ, David W, Wolfsen HC (January 2013). “Photodynamic Therapy for Barrett’s Esophagus and Esophageal Carcinoma”. Clinical Endoscopy. 46 (1): 30–37. doi:10.5946/ce.2013.46.1.30. PMC 3572348. PMID 23423151.]} Hyperthermia may also occur.^{[Tetard MC, Vermandel M, Mordon S, Lejeune JP, Reyns N (September 2014). “Experimental use of photodynamic therapy in high grade gliomas: a review focused on 5-aminolevulinic acid” (PDF). Photodiagnosis and Photodynamic Therapy. 11 (3): 319–330. doi:10.1016/j.pdpdt.2014.04.004. PMID 24905843.]} Deaths have also resulted.^{[Qumseya BJ, David W, Wolfsen HC (January 2013). “Photodynamic Therapy for Barrett’s Esophagus and Esophageal Carcinoma”. Clinical Endoscopy. 46 (1): 30–37. doi:10.5946/ce.2013.46.1.30. PMC 3572348. PMID 23423151.]}
    • In non-photosynthetic eukaryotes such as animals, fungi, and protozoa, as well as the class Alphaproteobacteria of bacteria, it is produced by the enzyme ALA synthase, from glycine and succinyl-CoA. This reaction is known as the Shemin pathway, which occurs in mitochondria.^{[Ajioka, James; Soldati, Dominique, eds. (September 13, 2007). “22”. Toxoplasma: Molecular and Cellular Biology (1 ed.). Taylor & Francis. p. 415. ISBN 9781904933342]}
    • In plants, algae, bacteria (except for the class Alphaproteobacteria) and archaea, it is produced from glutamic acid via glutamyl-tRNA and glutamate-1-semialdehyde. The enzymes involved in this pathway are glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde 2,1-aminomutase. This pathway is known as the C5 or Beale pathway.^{[Beale SI (August 1990). “Biosynthesis of the Tetrapyrrole Pigment Precursor, delta-Aminolevulinic Acid, from Glutamate”. Plant Physiology. 93 (4): 1273–1279. doi:10.1104/pp.93.4.1273. PMC 1062668. PMID 16667613.][Willows, R.D. (2004). “Chlorophylls”. In Goodman, Robert M. Encyclopaedia of Plant and Crop Science. Marcel Dekker. pp. 258–262. ISBN 0-8247-4268-0]} In most plastid-containing species, glutamyl-tRNA is encoded by a plastid gene, and the transcription, as well as the following steps of C5 pathway, take place in plastids.^{[Biswal, Basanti; Krupinska, Karin; Biswal, Udaya, eds. (2013). Plastid Development in Leaves during Growth and Senescence (Advances in Photosynthesis and Respiration). Dordrecht: Springer. p. 508. ISBN 9789400757233]}
    • Importance in humans
      • Activation of mitochondria
      • In humans, 5ALA is a precursor to heme.^{[Gardener LC, Cox TM (1988). “Biosynthesis of heme in immature erythroid cells”. The Journal of Biological Chemistry. 263: 6676–6682. doi:10.1016/S0021-9258(18)68695-8.]} Biosynthesized, 5ALA goes through a series of transformations in the cytosol and finally gets converted to Protoporphyrin IX inside the mitochondria.^{[Malik Z, Djaldetti M (June 1979). “5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells”. Cell Differentiation. 8 (3): 223–233. doi:10.1016/0045-6039(79)90049-6. PMID 288514.][Olivo M, Bhuvaneswari R, Keogh I (July 2011). “Advances in bio-optical imaging for the diagnosis of early oral cancer”. Pharmaceutics. 3 (3): 354–378. doi:10.3390/pharmaceutics3030354. PMC 3857071. PMID 24310585.]} This protoporphyrin molecule chelates with iron in presence of enzyme ferrochelatase to produce Heme.^{[Malik Z, Djaldetti M (June 1979). “5-Aminolevulinic acid stimulation of porphyrin and hemoglobin synthesis by uninduced Friend erythroleukemic cells”. Cell Differentiation. 8 (3): 223–233. doi:10.1016/0045-6039(79)90049-6. PMID 288514.][Olivo M, Bhuvaneswari R, Keogh I (July 2011). “Advances in bio-optical imaging for the diagnosis of early oral cancer”. Pharmaceutics. 3 (3): 354–378. doi:10.3390/pharmaceutics3030354. PMC 3857071. PMID 24310585.]}
      • Heme increases the mitochondrial activity thereby helping in activation of respiratory system Krebs Cycle and Electron Transport Chain^{[Ogura S, Maruyama K, Hagiya Y, Sugiyama Y, Tsuchiya K, Takahashi K, et al. (March 2011). “The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver”. BMC Research Notes. 4 (4): 66. doi:10.1186/1756-0500-4-66. PMC 3068109. PMID 21414200.]} leading to formation of adenosine triphosphate (ATP) for adequate supply of energy to the body.^{[Ogura S, Maruyama K, Hagiya Y, Sugiyama Y, Tsuchiya K, Takahashi K, et al. (March 2011). “The effect of 5-aminolevulinic acid on cytochrome c oxidase activity in mouse liver”. BMC Research Notes. 4 (4): 66. doi:10.1186/1756-0500-4-66. PMC 3068109. PMID 21414200.]}
      • Accumulation of Protoporphyrin IX
      • Cancer cells lack or have reduced ferrochelatase activity and this results in accumulation of Protoporphyrin IX, a fluorescent substance that can easily be visualized.^{[Wagnières, G.., Jichlinski, P., Lange, N., Kucera, P., Van den Bergh, H. (2014). Detection of Bladder Cancer by Fluorescence Cystoscopy: From Bench to Bedside – the Hexvix Story. Handbook of Photomedicine, 411-426.]}
      • Induction of Heme Oxygenase-1 (HO-1)
      • Excess heme is converted in macrophages to Biliverdin and ferrous ions by the enzyme HO-1. Biliverdin formed further gets converted to Bilirubin and carbon monoxide.^{[Loboda A, Damulewicz M, Pyza E, Jozkowicz A, Dulak J (September 2016). “Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism”. Cellular and Molecular Life Sciences. 73 (17): 3221–3247. doi:10.1007/s00018-016-2223-0. PMC 4967105. PMID 27100828.]} Biliverdin and Bilirubin are potent anti oxidants and regulate important biological processes like inflammation, apoptosis, cell proliferation, fibrosis and angiogenesis.^{[Loboda A, Damulewicz M, Pyza E, Jozkowicz A, Dulak J (September 2016). “Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism”. Cellular and Molecular Life Sciences. 73 (17): 3221–3247. doi:10.1007/s00018-016-2223-0. PMC 4967105. PMID 27100828.]}
    • Plants
      • In plants, production of 5-ALA is the step on which the speed of synthesis of chlorophyll is regulated.^{[Von Wettstein D, Gough S, Kannangara CG (July 1995). “Chlorophyll Biosynthesis”. The Plant Cell. 7 (7): 1039–1057. doi:10.1105/tpc.7.7.1039. PMC 160907. PMID 12242396.]} Plants that are fed by external 5-ALA accumulate toxic amounts of chlorophyll precursor, protochlorophyllide, indicating that the synthesis of this intermediate is not suppressed anywhere downwards in the chain of reaction. Protochlorophyllide is a strong photosensitizer in plants.^{[Kotzabasis K, Senger H (1990). “The influence of 5-aminolevulinic acid on protochlorophyllide and protochlorophyll accumulation in dark-grown Scenedesmus”. Z. Naturforsch. 45 (1–2): 71–73. doi:10.1515/znc-1990-1-212. S2CID 42965243.]} Controlled spraying of 5-ALA at lower doses (up to 150 mg/L) can however help protect plants from stress and encourage growth.^{[Kosar F, Akram NA, Ashraf M (January 2015). “Exogenously-applied 5-aminolevulinic acid modulates some key physiological characteristics and antioxidative defense system in spring wheat (Triticum aestivum L.) seedlings under water stress”. South African Journal of Botany. 96: 71–77. doi:10.1016/j.sajb.2014.10.015.]}
    • See also Lipoic acid
      • Lipoic acid (LA), also known as α-lipoic acid, alpha-lipoic acid (ALA) and thioctic acid, is an organosulfur compound derived from caprylic acid (octanoic acid). ALA is made in animals normally, and is essential for aerobic metabolism. It is also manufactured and is available as a dietary supplement in some countries where it is marketed as an antioxidant, and is available as a pharmaceutical drug in other countries. Lipoate is the conjugate base of lipoic acid, and the most prevalent form of LA under physiological conditions. Only the (R)-(+)-enantiomer (RLA) exists in nature and is essential for aerobic metabolism because RLA is an essential cofactor of many enzyme complexes. Lipoic acid (LA), also known as α-lipoic acid,^{[“Lipoic acid”. Micronutrient Information Center, Linus Pauling Institute, Oregon State University, Corvallis. 1 January 2019. Retrieved 5 November 2019][Shay, KP; Moreau, RF; Smith, EJ; Hagen, TM (June 2008). “Is alpha-lipoic acid a scavenger of reactive oxygen species in vivo? Evidence for its initiation of stress signaling pathways that promote endogenous antioxidant capacity”. IUBMB Life. 60 (6): 362–7. doi:10.1002/iub.40. PMID 18409172. S2CID 33008376.]} alpha-lipoic acid (ALA), and thioctic acid^{[Reljanovic, M; Reichel, G; Rett, K; Lobisch, M; et al. (September 1999). “Treatment of diabetic polyneuropathy with the antioxidant thioctic acid (alpha-lipoic acid): A two year multicenter randomized double-blind placebo-controlled trial (ALADIN II). Alpha Lipoic Acid in Diabetic Neuropathy”. Free Radical Research. 31 (3): 171–9. doi:10.1080/10715769900300721. PMID 10499773.]} is an organosulfur compound derived from octanoic acid. LA contains two sulfur atoms (at C6 and C8) connected by a disulfide bond and is thus considered to be oxidized although either sulfur atom can exist in higher oxidation states.^{[“Lipoic acid”. Micronutrient Information Center, Linus Pauling Institute, Oregon State University, Corvallis. 1 January 2019. Retrieved 5 November 2019.]} The carbon atom at C6 is chiral and the molecule exists as two enantiomers (R)-(+)-lipoic acid (RLA) and (S)-(-)-lipoic acid (SLA) and as a racemic mixture (R/S)-lipoic acid (R/S-LA). LA appears physically as a yellow solid and structurally contains a terminal carboxylic acid and a terminal dithiolane ring. For use in dietary supplement materials and compounding pharmacies, the USP established an official monograph for R/S-LA.^{[USP32-NF27. p. 1042.][ “Unavailable First-Time Official USP Reference Standards” (PDF). Pharmacopeial Forum. USP. 35: 26. February 2009. Archived (PDF) from the original on 5 March 2022. Retrieved 13 January 2023.]}
      • Biological function
        • Lipoic acid is a cofactor for five enzymes or classes of enzymes: pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, the glycine cleavage system, branched chain keto acid dehydrogenase, and the alpha-oxo(keto)adipate dehydrogenase. The first two are critical to the citric acid cycle. The GCS regulates glycine concentrations.^{[Cronan, John E. (2020). “Progress in the Enzymology of the Mitochondrial Diseases of Lipoic Acid Requiring Enzymes”. Frontiers in Genetics. 11: 510. doi:10.3389/fgene.2020.00510. PMC 7253636. PMID 32508887]}
• cytosolic:
  • Porphobilinogen
    • Porphobilinogen (PBG) is an organic compound that occurs in living organisms as an intermediate in the biosynthesis of porphyrins, which include critical substances like hemoglobin and chlorophyll.^{[Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb221. ISBN 978-0470048672.]} The structure of the molecule can be described as molecule of pyrrole with sidechains substituted for hydrogen atoms at positions 2, 3 and 4 in the ring (1 being the nitrogen atom); respectively, an aminomethyl group −CH₂−NH₂, an acetic acid (carboxymethyl) group −CH₂−COOH, and a propionic acid (carboxyethyl) group −CH₂−CH₂−COOH. In the first step of the porphyrin biosynthesis pathway, porphobilinogen is generated from aminolevulinate (ALA) by the enzyme ALA dehydratase. In the typical porphyrin biosynthesis pathway, four molecules of porphobilinogen are concatenated by carbons 2 and 5 of the pyrrole ring (adjacent to the nitrogen atom) into hydroxymethyl bilane by the enzyme porphobilinogen deaminase, also known as hydroxymethylbilane synthase. Acute intermittent porphyria causes an increase in urinary porphobilinogen.^{[ Aarsand, AK; Petersen PH; Sandberg S (April 2006). “Estimation and application of biological variation of urinary delta-aminolevulinic acid and porphobilinogen in healthy individuals and in patients with acute intermittent porphyria”. Clinical Chemistry. 52 (4): 650–656. doi:10.1373/clinchem.2005.060772. PMID 16595824.]}
  • Hydroxymethylbilane
    • Hydroxymethylbilane, also known as preuroporphyrinogen, is an organic compound that occurs in living organisms during the synthesis of porphyrins, a group of critical substances that include haemoglobin, myoglobin, and chlorophyll. The name is often abbreviated as HMB. The compound is a substituted bilane, a chain of four pyrrole rings interconnected by methylene bridges −CH₂−. The chain starts with a hydroxymethyl group −CH₂−OH and ends with an hydrogen, in place of the respective methylene bridges. The other two carbon atoms of each pyrrole cycle are connected to an acetic acid group −CH₂−COOH and a propionic acid group −CH₂−CH₂−COOH, in that order. ^{[Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb221. ISBN 978-0470048672.]} The compound is generated from four molecules of porphobilinogen by the enzyme porphobilinogen deaminase. The enzyme uroporphyrinogen III synthase closes the chain to form a porphyrinogen a class of compounds with the hexahydroporphine macrocycle; specifically, uroporphyrinogen III. In the absence of the enzyme, the compound undergoes spontaneous cyclization and becomes uroporphyrinogen I
      • In organic chemistry, bilane is a compound with the formula C₁₉H₂₀N₄ or [(C₄H₄N)−CH₂−(C₄H₃N)−]₂CH₂. It is a tetrapyrrole, a class of compounds with four independent pyrrole rings. Specifically, the molecule can be described as four pyrrole molecules C₄H₅N connected in an open chain by three methylene bridges −CH₂− at carbons adjacent to the nitrogens, replacing the respective hydrogens.^{[ Gerard P. Moss (1988). “Nomenclature of Tetrapyrroles. Recommendations 1986”. European Journal of Biochemistry. 178 (2): 277–328. doi:10.1111/j.1432-1033.1988.tb14453.x. PMID 3208761.]}
      • The name is also used for the class of compounds formally derived from bilane proper by replacement of some additional hydrogen atoms by various functional groups. Natural bilanes usually have side chains substituted on the two carbons in each pyrrole ring that are not adjacent to the nitrogens. Artificial bilanes may be substituted on the bridging carbons (called meso positions).^{[Lindsey, J. S. (2010). “Synthetic Routes to meso-Patterned Porphyrins”. Accounts of Chemical Research. 43 (2): 300–311. doi:10.1021/ar900212t. PMID 19863076.]}
      • The parent (unsubstituted) bilane is difficult to prepare and unstable,^{[Claudia Ryppa, Mathias O. Senge, Sabine S. Hatscher, Erich Kleinpeter, Philipp Wacker, Uwe Schilde, and Arno Wiehe (2005): “Synthesis of Mono‐ and Disubstituted Porphyrins: A‐ and 5,10‐A₂‐Type Systems”. Chemistry, A European Journal, volume 11, issue 11, pages 3427-3442. doi:10.1002/chem.20050000]} but substituted derivatives are synthesized by most living organisms as intermediates in the synthesis of natural porphyrins. Substituted bilanes may also be the starting point for the synthesis of artificial porphyrins.^{[Lindsey, J. S. (2010). “Synthetic Routes to meso-Patterned Porphyrins”. Accounts of Chemical Research. 43 (2): 300–311. doi:10.1021/ar900212t. PMID 19863076.][}Claudia Ryppa, Mathias O. Senge, Sabine S. Hatscher, Erich Kleinpeter, Philipp Wacker, Uwe Schilde, and Arno Wiehe (2005): “Synthesis of Mono‐ and Disubstituted Porphyrins: A‐ and 5,10‐A₂‐Type Systems”. Chemistry, A European Journal, volume 11, issue 11, pages 3427-3442. doi:10.1002/chem.20050000^]
      • Upon treatment with aldehydes, bilanes may cyclize to give porphyrinogens and various open or closed oligomers and polymers.^{[Lindsey, J. S. (2010). “Synthetic Routes to meso-Patterned Porphyrins”. Accounts of Chemical Research. 43 (2): 300–311. doi:10.1021/ar900212t. PMID 19863076.]} In living organisms, the biosynthesis of all natural porphyrins proceeds through the bilane preuroporphyrinogen, which is produced from four molecules of the monomer porphobilinogen, and then converted to the closed tetrapyrrole uroporphyrinogen III (or, in certain metabolic disorders, into uroporphyrinogen I). Also, the catabolism of hemoglobin in humans produces bilirubin, another linear tetrapyrrole that is a partially oxidized bilane.
  • Uroporphyrinogen III
    • Uroporphyrinogen III is a tetrapyrrole, the first macrocyclic intermediate in the biosynthesis of heme, chlorophyll, vitamin B12, and siroheme. It is a colorless compound, like other porphyrinogens.^{[Dalton, J (1969). “Formation of the Macrocyclic Ring in Tetrapyrrole Biosynthesis”. Nature. 223 (5211): 1151–1153. Bibcode:1969Natur.223.1151D. doi:10.1038/2231151a0. PMID 5810686. S2CID 4177167.]} The molecular structure of uroporphyrinogen III can be described as a hexahydroporphine core, where each pyrrole ring has the hydrogen atoms on its two outermost carbons replaced by an acetic acid group (−CH₂−COOH, “A”) and a propionic acid group (−CH₂−CH₂−COOH, “P”). The groups are attached in an asymmetric way: going around the macrocycle, the order is AP-AP-AP-PA.
      • Siroheme (or sirohaem) is a heme-like prosthetic group at the active sites of some enzymes to accomplish the six-electronreduction of sulfur and nitrogen.^{[Matthew J. Murphy; et al. (1974). “Siroheme: A New Prosthetic Group Participating in Six-Electron Reduction Reactions Catalyzed by Both Sulfite and Nitrite Reductases”. PNAS. 71 (3): 612–616. Bibcode:1974PNAS…71..612M. doi:10.1073/pnas.71.3.612. PMC 388061. PMID 4595566.]} It is a cofactor at the active site of sulfite reductase, which plays a major role in sulfur assimilation pathway, converting sulfite into sulfide, which can be incorporated into the organic compound homocysteine.^{[Dominique Thomas; Yolande Surdin-Kerjan (1997). “Metabolism of sulfur amino acids in Saccharomyces cerevisiae”. Microbiology and Molecular Biology Reviews. 61 (4): 503–532. doi:10.1128/mmbr.61.4.503-532.1997. PMC 232622. PMID 9409150.]} Like all tetrapyrroles, the macrocyclic ligand in siroheme is derived from uroporphyrinogen III. This porphyrinogen is methylated at two adjacent pyrrole rings to give dihydrosirohydrochlorin, which is subsequently oxidized to give sirohydrochlorin. A ferrochelatase then inserts iron into the macrocycle to give siroheme.^{[Kaushik Saha, Michaël Moulin, Alison G. Smith (2009). “Tetrapyrroles in Plants: Chemical Biology of Metal Insertion and Removal”. Wiley Encyclopedia of Chemical Biology. Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb454. ISBN 978-0470048672.]}
      • See also Ferredoxin-nitrite reductase, Hydrogensulfite reductase, Nitrite reductase (NAD(P)H)
    • The conversion entails a reversal of the last pyrrole unit (thus swapping the acetic and propionic acid groups) and a condensation reaction that closes the macrocycle by eliminating the final hydroxyl −OH with a hydrogen atom of the first ring.
    • In the biosynthesis of hemes and chlorophylls, uroporphyrinogen III is converted into coproporphyrinogen III by the enzyme uroporphyrinogen III decarboxylase. In the biosynthesis of sirohemes, uroporphyrinogen III is converted by two methyl transferases to dihydrosirohydrochlorin, which is subsequently oxidized sirohydrochlorin, a precursor to the siroheme prosthetic group. If uroporphyrinogen-III synthase is not present or inactive, the hydroxymethylbilane will spontaneously cyclise into the structural isomer uroporphyrinogen I, which differs from the III isomer in that the acetic acid (“A”) and propionic acid (“P”) groups are arranged in a rotationally symmetric order, AP-AP-AP-AP. In this case, the next step produced coproporphyrinogen I, which accumulates — leading to the pathological condition congenital erythropoietic porphyria^{[ S. Sassa and A. Kappas (2000): “Molecular aspects of the inherited porphyrias”. Journal of Internal Medicine, volume 247, issue 2, pages 169-178. doi:10.1046/j.1365-2796.2000.00618.x]}
      • See also Uroporphyrinogen
        • Uroporphyrinogens are cyclic tetrapyrroles with four propionic acid groups (“P” groups) and four acetic acid groups (“A” groups). There are four forms, which vary based upon the arrangements of the “P” and “A” groups (in clockwise order): In the “I” variety (i.e. uroporphyrinogen I), the order repeats four times: AP-AP-AP-AP, In the “III” variety (i.e. uroporphyrinogen III), the fourth is reversed: AP-AP-AP-PA. This is the most common form. In the synthesis of porphyrin, it is created from the linear tetrapyrrole hydroxymethylbilane by the enzyme uroporphyrinogen III synthase, and is further converted into coproporphyrinogen III by the enzyme uroporphyrinogen III decarboxylase. The “II” and “IV” varieties can be created synthetically, but do not appear in nature.
  • Coproporphyrinogen III
    • Coproporphyrinogen III is a metabolic intermediate in the biosynthesis of many compounds that are critical for living organisms, such as hemoglobin and chlorophyll. It is a colorless solid. The compound is a porphyrinogen, a class of compounds characterized by a hexahydroporphine core with various side chains. The coproporphyrinogens have the outermost hydrogen atoms of the core replaced by four methyl groups −CH₃ (M) and four propionic acid groups −CH₂−CH₂−COOH (P). In coproporphyrogen III, the order around the outer ring is MP-MP-MP-PM. For comparison, coproporphyrinogen I has them in the sequence MP-MP-MP-MP. heme. In the main porphyrin biosynthesis pathway, coproporphyrinogen III is derived from uroporphyrinogen III by the action of the enzyme uroporphyrinogen III decarboxylase. The conversion entails four decarboxylations, which turn the four acetic acid groups −CH₂−COOH into methyl groups −CH₃, with release of four carbon dioxide molecules.^{[ Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb221. ISBN 978-0470048672.][Sassa, S.; Kappas, A. (2000). “Molecular aspects of the inherited porphyrias”. Journal of Internal Medicine. 247 (2): 169–78. doi:10.1046/j.1365-2796.2000.00618.x. PMID 10692079. S2CID 36820694.]} Coproporphyrinogen III is further used as a substrate for the enzyme coproporphyrinogen III oxidase which oxidizes and further decarboxylates it to protoporphyrinogen IX.
• late mitochondrial:
  • Protoporphyrinogen IX
    • Protoporphyrinogen IX is an organic chemical compound which is produced along the synthesis of porphyrins, a class of critical biochemicals that include hemoglobin and chlorophyll. It is a direct precursor of protoporphyrin IX. The compound is a porphyrinogen, meaning that it has a non-aromatic hexahydroporphine core, which will be oxidized to a porphine core in later stages of the heme synthesis. Like most porphyrinogens, it is colorless.^{[citation needed]} The compound is synthesized in most organisms from coproporphyrinogen III by the enzyme coproporphyrinogen oxidase. The process entails conversion of two of four propionic acid groups to vinyl groups. In coproporphyrinogen III, the substituents on the pyrrole rings have the arrangement MP-MP-MP-PM, where M and P are methyl and propionic acid, respectively. In protoporphyrinogen IX, the sequence becomes MV-MV-MP-PM, where V is vinyl. By the action of protoporphyrinogen oxidase, protoporphyrinogen IX is later converted into protoporphyrin IX, the first colored tetrapyrrole in the biosynthesis of hemes.^{[Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb221. ISBN 978-0470048672.]}
    • See also: Protoporphyrinogen oxidase
      • Protoporphyrinogen oxidase is responsible for the seventh step in biosynthesis of protoporphyrin IX. This porphyrin is the precursor to hemoglobin, the oxygen carrier in animals, and chlorophyll, the dye in plants. The enzyme catalyzes the dehydrogenation (removal of hydrogen atoms) of protoporphyrinogen IX (the product of the sixth step in the production of heme) to form protoporphyrin IX. One additional enzyme must modify protoporphyrin IX before it becomes heme. Inhibition of this enzyme is a strategy used in certain herbicides.
      • Clinical significance
        • Variegate porphyria is caused by mutations in the PPOX gene. More than 100 mutations that can cause variegate porphyria have been identified in the PPOX gene. One mutation, a substitution of the amino acid tryptophan for arginine at position 59 (also written as Arg59Trp or R59W), is found in about 95 percent of South African families with variegate porphyria. Mutations in the PPOX gene reduce the activity of the enzyme made by the gene, allowing byproducts of heme production to build up in the body. This buildup, in combination with nongenetic factors (such as certain drugs, alcohol and dieting), causes this type of porphyria.
      • Inhibitors as herbicides
        • Inhibition of protoporphyrinogen oxidase is a mechanism of action for several commercial herbicides including the nitrophenyl ethers acifluorfen and fomesafen and the pyrimidinediones butafenacil and saflufenacil. The visible symptoms of treatment are chlorosis and desiccation. The damage is caused by an accumulation of protoporphyrin IX in the plant cells by inhibiting protox within the tetrapyrrole biosynthesis pathway.^{[Brzezowski P, Ksas B, Havaux M, Grimm B, Chazaux M, Peltier G, et al. (2019-05-03). “The function of PROTOPORPHYRINOGEN IX OXIDASE in chlorophyll biosynthesis requires oxidised plastoquinone in Chlamydomonas reinhardtii“. Communications Biology. 2 (1): 159. doi:10.1038/s42003-019-0395-5. PMC 6499784. PMID 31069268.]} This is a potent photosensitizer which activates oxygen, leading to lipid peroxidation. Both light and oxygen are required for this process to kill the plant.^{[Dayan FE, Reddy KN, Duke SO (1999). “Structure-Activity Relationships of Diphenyl Ethers and Other Oxygen-Bridged Protoporphyrinogen Oxidase Inhibitors”. Peroxidizing Herbicides. pp. 141–161. doi:10.1007/978-3-642-58633-0_5. ISBN 978-3-642-63674-5.][Nagano E (1999). “Herbicidal Efficacy of Protoporphyrinogen Oxidase Inhibitors”. Peroxidizing Herbicides. pp. 293–302. doi:10.1007/978-3-642-58633-0_11. ISBN 978-3-642-63674-5.][Dayan FE, Duke SO (2010). “Protoporphyrinogen Oxidase-Inhibiting Herbicides”. Hayes’ Handbook of Pesticide Toxicology. pp. 1733–1751. doi:10.1016/B978-0-12-374367-1.00081-1. ISBN 9780123743671.]}
      • The following genes encode enzymes that catalyze the various steps in the heme biosynthetic pathway:
        • ALAD: aminolevulinate, delta-, dehydratase
        • ALAS1: aminolevulinate, delta-, synthase 1
        • ALAS2: aminolevulinate, delta-, synthase 2 (sideroblastic/hypochromic anemia)
        • CPOX: coproporphyrinogen oxidase
        • FECH: ferrochelatase (protoporphyria)
          • Medlock AE, Carter M, Dailey TA, Dailey HA, Lanzilotta WN. Product release rather than chelation determines metal specificity for ferrochelatase. J Mol Biol. 2009 Oct 23;393(2):308-19. doi: 10.1016/j.jmb.2009.08.042. Epub 2009 Aug 22. PMID: 19703464; PMCID: PMC2771925.
        • HMBS: hydroxymethylbilane synthase
        • PPOX: protoporphyrinogen oxidase
        • UROD: uroporphyrinogen decarboxylase
        • UROS: uroporphyrinogen III synthase (congenital erythropoietic porphyria)
      • See also Porphyrin
  • Protoporphyrin IX
    • Protoporphyrin IX is an organic compound, classified as a porphyrin, that plays an important role in living organisms as a precursor to other critical compounds like heme (hemoglobin) and chlorophyll. It is a deeply colored solid that is not soluble in water. The name is often abbreviated as PPIX. Protoporphyrin IX contains a porphine core, a tetrapyrrole macrocycle with a marked aromatic character. Protoporphyrin IX is essentially planar, except for the N-H bonds that are bent out of the plane of the rings, in opposite (trans) directions.^{[Winslow S. Caughey, James A. Ibers (1977). “Crystal and Molecular Structure of the Free Base Porphyrin, Protoporphyrin IX Dimethyl Ester”. J. Am. Chem. Soc. 99 (20): 6639–6645. doi:10.1021/ja00462a027. PMID 19518.]} The general term protoporphyrin refers to porphine derivatives that have the outer hydrogen atoms in the four pyrrole rings replaced by other functional groups. The prefix proto often means ‘first’ in science nomenclature (such as carbon protoxide), hence Hans Fischer is thought to have coined the name protoporphyrin as the first class of porphyrins.^{[Vicente, Maria da G.H.; Smith, Kevin M. (2014). “Syntheses and Functionalizations of Porphyrin Macrocycles”. Current Organic Synthesis. 11 (1): 3–28. doi:10.2174/15701794113106660083. ISSN 1570-1794. PMC 4251786. PMID 25484638]} Fischer described iron-deprived heme becoming the “proto-” porphyrin, particularly in reference to Hugo Kammerer’s porphyrin.^{[Fischer, Hans (1930). “On haemin and the relationships between haemin and chlorophyll” (PDF). Nobel Prize.][With, Torben K. (1980-01-01). “A short history of porphyrins and the porphyrias”. International Journal of Biochemistry. 11 (3–4): 189–200. doi:10.1016/0020-711X(80)90219-0. ISSN 0020-711X. PMID 6993245]} In modern times, ‘proto-‘ specifies a porphyrin species bearing methyl, vinyl, and carboxyethyl/propionate side groups.^{[Neves, Ana Carolina de Oliveira; Galván, Ismael (2020). “Models for human porphyrias: Have animals in the wild been overlooked?”. BioEssays. 42 (12): 2000155. doi:10.1002/bies.202000155. ISSN 1521-1878. PMID 33155299. S2CID 226269267.]} Fischer also generated the Roman numeral naming system which includes 15 protoporphyrin analogs, the naming system is not systematic however.^{[Moss, G. P. (1988-12-15). “Nomenclature of tetrapyrroles. Recommendations 1986 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN)”. European Journal of Biochemistry. 178 (2): 277–328. doi:10.1111/j.1432-1033.1988.tb14453.x. ISSN 0014-2956. PMID 3208761.]} An alternative name for heme is iron protoporphyrin IX (iron PPIX). PPIX contains four methyl groups −CH₃ (M), two vinyl groups −CH=CH₂ (V), and two propionic acid groups −CH₂−CH₂−COOH (P). The suffix “IX” indicates that these chains occur in the circular order MV-MV-MP-PM around the outer cycle at the following respective positions: c2,c3-c7,c8-c12,c13-c17,c18.^{[Moss, G. P. (1988-12-15). “Nomenclature of tetrapyrroles. Recommendations 1986 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN)”. European Journal of Biochemistry. 178 (2): 277–328. doi:10.1111/j.1432-1033.1988.tb14453.x. ISSN 0014-2956. PMID 3208761.]} The methine bridges of PPIX are named alpha (c5), beta (c10), gamma (c15), and delta (c20). In the context of heme, metabolic biotransformation by heme oxygenase results in the selective opening of the alpha-methine bridge to form biliverdin/bilirubin. In this case, the resulting bilin carries the suffix IXα which indicates the parent molecule was protoporphyrin IX cleaved at the alpha position. Non-enzymatic oxidation may result in the ring opening at other bridge positions.^{[Berk, Paul D.; Berlin, Nathaniel I. (1977). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health]} The use of Greek letters in this context originates from the pioneering work of Georg Barkan in 1932.^[Barkan, Georg; Schales, Otto (1938). “A Hæmoglobin from Bile Pigment”. Nature. 142 (3601): 836–837. Bibcode:1938Natur.142..836B. doi:10.1038/142836b0. ISSN 1476-4687. S2CID 4073510.^]
    • Properties
      • When UV light is shone on the compound, it fluoresces with a bright red color.
      • It Is also the component in egg shells that give them their characteristic brown color.^{[Sachar, M.; Anderson, K. E.; Ma, X. (2016). “Protoporphyrin IX: The Good, the Bad, and the Ugly”. Journal of Pharmacology and Experimental Therapeutics. 356 (2): 267–275. doi:10.1124/jpet.115.228130. PMC 4727154. PMID 26588930.]}
    • Natural occurrence
      • The compound is encountered in nature in the form of complexes where the two inner hydrogen atoms are replaced by a divalent metal cation. When complexed with an iron(II) (ferrous) cation Fe²⁺, the molecule is called heme. Hemes are prosthetic groups in some important proteins. These heme-containing proteins include hemoglobin, myoglobin, and cytochrome c. Complexes can also be formed with other metal ions, such as zinc.^{[Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb221. ISBN 978-0470048672.]}
    • Biosynthesis
      • Main article: Porphyrin § Synthesis
      • The compound is synthesized from acyclic precursors via a mono-pyrrole (porphobilinogen) then a tetrapyrrole (a porphyrinogen, specifically uroporphyrinogen III). This precursor is converted to protoporphyrinogen IX, which is oxidized to protoporphyrin IX.^{[Paul R. Ortiz de Montellano (2008). “Hemes in Biology”. Wiley Encyclopedia of Chemical Biology. John Wiley & Sons. doi:10.1002/9780470048672.wecb221. ISBN 978-0470048672.]} The last step is mediated by the enzyme protoporphyrinogen oxidase.
      • In the biosynthesis of those molecules, the metal cation is inserted into protoporphyrin IX by enzymes called chelatases. For example, ferrochelatase converts the compound into heme B (i.e. Fe-protoporphyrin IX or protoheme IX). In chlorophyll biosynthesis, the enzyme magnesium chelatase converts it into Mg-protoporphyrin IX.
    • Described metalloprotoporphyrin IX derivatives
      • Palepron is the disodium salt of protoporphyrin IX.^{[PubChem. “Protoporphyrin disodium”. pubchem.ncbi.nlm.nih.gov. Retrieved 2021-04-15.]}
    • History
      • Laidlaw may have first isolated PPIX in 1904.^{[With, Torben K. (1980-01-01). “A short history of porphyrins and the porphyrias”. International Journal of Biochemistry. 11 (3–4): 189–200. doi:10.1016/0020-711X(80)90219-0. ISSN 0020-711X. PMID 6993245]}
    • See also: Carbon monoxide-releasing molecules, Heme oxygenase, Biosynthesis of chlorophylls, Biosynthesis of hemes

Heme degradation and excretion

• Breakdown of heme
  • spleen:
    • Heme → Biliverdin → Bilirubin
      • Biliverdin (from the Latin for green bile) is a green tetrapyrrolic bilepigment, and is a product of hemecatabolism.^{[Boron W, Boulpaep E. Medical Physiology: a cellular and molecular approach, 2005. 984-986. Elsevier Saunders, United States. ISBN 1-4160-2328-3]} 
      • It is the pigment responsible for a greenish color sometimes seen in bruises.^{[Mosqueda, L; Burnight, K; Liao, S (2005). “The Life Cycle of Bruises in Older Adults”. Journal of the American Geriatrics Society. 53 (8): 1339–1343. doi:10.1111/j.1532-5415.2005.53406.x. PMID 16078959. S2CID 12394659.]}
      • Biliverdin results from the breakdown of the heme moiety of hemoglobin in erythrocytes. Macrophages break down senescent erythrocytes and break the heme down into biliverdin along with hemosiderin, in which biliverdin normally rapidly reduces to free bilirubin.^{[Boron W, Boulpaep E. Medical Physiology: a cellular and molecular approach, 2005. 984-986. Elsevier Saunders, United States. ISBN 1-4160-2328-3][Seyfried, H; Klicpera, M; Leithner, C; Penner, E (1976). “Bilirubin metabolism (author’s transl)”. Wiener Klinische Wochenschrift. 88 (15): 477–82. PMID 793184.]}
      • Biliverdin is seen briefly in some bruises as a green color. In bruises, its breakdown into bilirubin leads to a yellowish color.^{[Mosqueda, L; Burnight, K; Liao, S (2005). “The Life Cycle of Bruises in Older Adults”. Journal of the American Geriatrics Society. 53 (8): 1339–1343. doi:10.1111/j.1532-5415.2005.53406.x. PMID 16078959. S2CID 12394659.]}
      • Role in disease
        • Biliverdin has been found in excess in the blood of humans suffering from hepatic diseases. Jaundice is caused by the accumulation of biliverdin or bilirubin (or both) in the circulatory system and tissues.^{[Boron W, Boulpaep E. Medical Physiology: a cellular and molecular approach, 2005. 984-986. Elsevier Saunders, United States. ISBN 1-4160-2328-3]} Jaundiced skin and sclera (whites of the eyes) are characteristic of liver failure.
      • Role in treatment of disease
        • While typically regarded as a mere waste product of heme breakdown, evidence that suggests that biliverdin – and other bile pigments – has a physiological role in humans has been mounting.^{[Bulmer, A. C.; Ried, K.; Blanchfield, J. T.; Wagner, K. H. (2008). “The anti-mutagenic properties of bile pigments”. Mutation Research. 658 (1–2): 28–41. doi:10.1016/j.mrrev.2007.05.001. PMID 17602853.][Ohrui, T.; Yasuda, H.; Yamaya, M.; Matsui, T.; Sasaki, H. (2003). “Transient relief of asthma symptoms during jaundice: a possible beneficial role of bilirubin”. The Tohoku Journal of Experimental Medicine. 199 (3): 193–196. doi:10.1620/tjem.199.193. PMID 12703664.]}
        • Bile pigments such as biliverdin possess significant anti-mutagenic and antioxidant properties and therefore, may fulfil a useful physiological function. Biliverdin and bilirubin have been shown to be potent scavengers of hydroperoxyl radicals.^{[Bulmer, A. C.; Ried, K.; Blanchfield, J. T.; Wagner, K. H. (2008). “The anti-mutagenic properties of bile pigments”. Mutation Research. 658 (1–2): 28–41. doi:10.1016/j.mrrev.2007.05.001. PMID 17602853.][Ohrui, T.; Yasuda, H.; Yamaya, M.; Matsui, T.; Sasaki, H. (2003). “Transient relief of asthma symptoms during jaundice: a possible beneficial role of bilirubin”. The Tohoku Journal of Experimental Medicine. 199 (3): 193–196. doi:10.1620/tjem.199.193. PMID 12703664.]} They have also been shown to inhibit the effects of polycyclic aromatic hydrocarbons, heterocyclic amines, and oxidants – all of which are mutagens. Some studies have found that people with higher concentration levels of bilirubin and biliverdin in their bodies have a lower frequency of cancer and cardiovascular disease.^{[Bulmer, A. C.; Ried, K.; Blanchfield, J. T.; Wagner, K. H. (2008). “The anti-mutagenic properties of bile pigments”. Mutation Research. 658 (1–2): 28–41. doi:10.1016/j.mrrev.2007.05.001. PMID 17602853.]} It has been suggested that biliverdin – as well as many other tetrapyrrolic pigments – may function as an HIV-1 protease inhibitor^{[McPhee, F.; Caldera, P. S.; Bemis, G. W.; McDonagh, A. F.; Kuntz, I. D.; Craik, C. S. (1996). “Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro”. The Biochemical Journal. 320 (Pt 2): 681–686. doi:10.1042/bj3200681. PMC 1217983. PMID 8973584.]} as well as having beneficial effects in asthma^{[Ohrui, T.; Yasuda, H.; Yamaya, M.; Matsui, T.; Sasaki, H. (2003). “Transient relief of asthma symptoms during jaundice: a possible beneficial role of bilirubin”. The Tohoku Journal of Experimental Medicine. 199 (3): 193–196. doi:10.1620/tjem.199.193. PMID 12703664.]} though further research is needed to confirm these results. There are currently no practical implications for using biliverdin in the treatment of any disease.
      • In non-human animals
        • Biliverdin is an important pigment component in avian egg shells, especially blue and green shells. Blue egg shells have a significantly higher concentration of biliverdin than brown egg shells.^{[Halepas, Steven; Hamchand, Randy; Lindeyer, Samuel E. D.; Brückner, Christian (2017). “Isolation of Biliverdin IXα, as its Dimethyl Ester, from Emu Eggshells”. Journal of Chemical Education. 94 (10): 1533–1537. Bibcode:2017JChEd..94.1533H. doi:10.1021/acs.jchemed.7b00449.]}
        • Research has shown that the biliverdin of egg shells is produced from the shell gland, rather than from the breakdown of erythrocytes in the blood stream,^{[citation needed]} although there is no evidence that the sources of the material are neither tetrapyrroles nor free haem from the blood plasma.^{[clarification needed][citation needed]}
        • Along with its presence in avian egg shells, other studies have also shown that biliverdin is present in the blue-green blood of many marine fish, the blood of tobacco hornworm, the wings of moth and butterfly, the serum and eggs of frogs, and the placenta of dogs.^{[Fang, LS; Bada, JL (1990). “The blue-green blood plasma of marine fish”. Comparative Biochemistry and Physiology B. 97 (1): 37–45. doi:10.1016/0305-0491(90)90174-R. PMID 2253479.]} With dogs this can lead, in extremely rare cases, to the birth of puppies with green fur; however, the green color fades out soon after birth.^{[ “These Puppies Were Born with Green Fur”.]} In the garfish (Belone belone) and related species, the bones are bright green because of biliverdin.^{[citation needed]} The green coloration of many grasshoppers and lepidopteran larvae is also due to biliverdin.^{[Shamim, G; Ranjan, S; Pandey, D; Ramani, R (2014). “Biochemistry and biosynthesis of insect pigments” (PDF). European Journal of Entomology. 111(2): 155. doi:10.14411/eje.2014.021. Retrieved 25 June 2023.]}
        • Biliverdin is also present in the green blood, muscles, bones, and mucosal lining of skinks of the genus Prasinohaema, found in New Guinea. It is uncertain whether this presence of biliverdin is an ecological or physiological adaptation of any kind. It has been suggested that accumulation of biliverdin might deter harmful infection by Plasmodium malaria parasites, although no statistically significant correlation has been established.^{[ Austin, C; Perkins, S (2006). “Parasites in a biodiversity hotspot: a survey of hematozoa and a molecular phyolgenetic analysis of plasmodium in New Guinea skinks”. Journal of Parasitology. 92 (4): 770–777. doi:10.1645/GE-693R.1. PMID 16995395. S2CID 1937837.]} The Cambodian frog, Chiromantis samkosensis, also exhibits this characteristic along with turquoise bones.^{[Lee Grismer, L.; Thy, Neang; Chav, Thou; Holden, Jeremy (2007). “A New Species of Chiromantis Peters 1854 (Anura: Rhacophoridae) from Phnom Samkos in the Northwestern Cardamom Mountains, Cambodia”. Herpetologica. 63 (3): 392–400. doi:10.1655/0018-0831(2007)63[392:ANSOCP]2.0.CO;2. S2CID 84472376.]}
      • In fluorescence imaging
        • In a complex with reengineered bacterial phytochrome, biliverdin has been employed as an IR-emitting chromophore for in vivo imaging.^{[X. Shu; et al. (2009). “Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome”. Science. 324 (5928): 804–807. Bibcode:2009Sci…324..804S. doi:10.1126/science.1168683. PMC 2763207. PMID 19423828][GSFilonov; Piatkevich, Kiryl D; Ting, Li-Min; Zhang, Jinghang; Kim, Kami; Verkhusha, Vladislav V; et al. (2011). “Bright and stable near infra-red fluorescent protein for in vivo imaging”. Nat Biotechnol. 29 (8): 757–761. doi:10.1038/nbt.1918. PMC 3152693. PMID 21765402.]} In contrast to fluorescent proteins which form their chromophore through posttranslational modifications of the polypeptide chain, phytochromes bind an external ligand (in this case, biliverdin), and successful imaging of the first bacteriophytochrome-based probe required addition of the exogenous biliverdin.^{[X. Shu; et al. (2009). “Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome”. Science. 324 (5928): 804–807. Bibcode:2009Sci…324..804S. doi:10.1126/science.1168683. PMC 2763207. PMID 19423828.]} Recent studies demonstrated that bacteriophytochrome-based fluorescent proteins with high affinity to biliverdin can be imaged in vivo utilizing endogenous ligand only and, thus, with the same ease as the conventional fluorescent proteins.^{[GSFilonov; Piatkevich, Kiryl D; Ting, Li-Min; Zhang, Jinghang; Kim, Kami; Verkhusha, Vladislav V; et al. (2011). “Bright and stable near infra-red fluorescent protein for in vivo imaging”. Nat Biotechnol. 29 (8): 757–761. doi:10.1038/nbt.1918. PMC 3152693. PMID 21765402.]} Advent of the second and further generations of the biliverdin-binding bacteriophytochrome-based probes should broaden the possibilities for the non-invasive in vivo imaging.
        • A new class of fluorescent protein was evolved from a cyanobacterial (Trichodesmium erythraeum) phycobiliprotein, α-allophycocyanin, and named small ultra red fluorescent protein (smURFP) in 2016. smURFP autocatalytically self-incorporates the chromophore biliverdin without the need of an external protein, known as a lyase.^{[Rodriguez, Erik A.; Tran, Geraldine N.; Gross, Larry A.; Crisp, Jessica L.; Shu, Xiaokun; Lin, John Y.; Tsien, Roger Y. (2016-08-01). “A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein”. Nature Methods. 13 (9): 763–9. doi:10.1038/nmeth.3935. ISSN 1548-7105. PMC 5007177. PMID 27479328.]} Jellyfish– and coral-derived fluorescent proteins require oxygen and produce a stoichiometric amount of hydrogen peroxide upon chromophore formation.^{[Tsien, Roger Y. (1998-01-01). “The Green Fluorescent Protein”. Annual Review of Biochemistry. 67 (1): 509–544. doi:10.1146/annurev.biochem.67.1.509. PMID 9759496.]} smURFP does not require oxygen or produce hydrogen peroxide and uses the chromophore biliverdin. smURFP has a large extinction coefficient (180,000 M⁻¹ cm⁻¹) and has a modest quantum yield (0.20), which makes it comparable biophysical brightness to eGFP and about 2-fold brighter than most red or far-red fluorescent proteins derived from coral. smURFP spectral properties are similar to the organic dye Cy5.^{[Rodriguez, Erik A.; Tran, Geraldine N.; Gross, Larry A.; Crisp, Jessica L.; Shu, Xiaokun; Lin, John Y.; Tsien, Roger Y. (2016-08-01). “A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein”. Nature Methods. 13 (9): 763–9. doi:10.1038/nmeth.3935. ISSN 1548-7105. PMC 5007177. PMID 27479328.]}
          • Cy5– Cyanines, also referred to as tetramethylindo(di)-carbocyanines^{[Kvach, Maksim V.; Ustinov, Alexey V.; Stepanova, Irina A.; Malakhov, Andrei D.; Skorobogatyi, Mikhail V.; Shmanai, Vadim V.; Korshun, Vladimir A. (2008). “A Convenient Synthesis of Cyanine Dyes: Reagents for the Labeling of Biomolecules”. European Journal of Organic Chemistry. 2008 (12): 2107–2117. doi:10.1002/ejoc.200701190. ISSN 1099-0690.]} are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic spectrum from near IR to UV.
          • Chemically, cyanines are a conjugated system between two nitrogen atoms; in each resonance structure, exactly one nitrogen atom is oxidized to an iminium. Typically, they form part of a nitrogenous heterocyclic system.^{[“Cyanine dyes”. The IUPAC Compendium of Chemical Terminology. 2014. doi:10.1351/goldbook.C01487.]}
          • The main application for cyanine dyes is in biological labeling. Nevertheless, there is a wide literature on both their synthesis and uses, and cyanines are common in some CD and DVD media.
          • Cyanines have been classified in many ways:^{[Kim, Eunha; Park, Seung Bum (2010). “Discovery of New Synthetic Dyes: Targeted Synthesis or Combinatorial Approach?”. In Demchenko, Alexander P. (ed.). Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design Volume 8 of Springer Series on Fluorescence. Berlin: Springer. p. 172. ISBN 9783642047022.]}
          • Streptocyanines or open chain cyanines:
          • R₂N⁺=CH[CH=CH]_n-NR₂(I)
          • Hemicyanines:
          • Aryl=N⁺=CH[CH=CH]_n-NR₂(II)
          • Closed chain cyanines:
          • Aryl=N⁺=CH[CH=CH]_n-N=Aryl (III)
          • Additionally, these classes are recognized:^{[Berneth, Horst (2008). “Methine Dyes and Pigments”. Ullmann’s Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a16_487.pub2.]}
          • Neutrocyanines:
          • R₂N⁺=CH[CH=CH]_n-CN and R₂N⁺=CH[CH=CH]_n-CHO
          • Merocyanines including spiropyrans and quinophthalones.
            • A spiropyran is a type of organic chemical compound, known for photochromic properties that provide this molecule with the ability of being used in medical and technological areas. Spiropyrans were discovered in the early twentieth century.^{[ Kortekaas L, Browne WR (June 2019). “The evolution of spiropyran: fundamentals and progress of an extraordinarily versatile photochrome”. Chemical Society Reviews. 48 (12): 3406–3424. doi:10.1039/C9CS00203K. PMID 31150035.]} However, it was in the middle twenties when Fisher and Hirshbergin observed their photochromic characteristics and reversible reaction. In 1952, Fisher and co-workers announced for the first time photochromism in spiropyrans. Since then, there have been many studies on photochromic compounds that have continued up to the present.^{[ Lukyanov BS, Lukyanova MB (2005). “Spiropyrans: Synthesis, Properties, and Application. A review”. Chemistry of Heterocyclic Compounds. 41 (3): 281–311. doi:10.1007/s10593-005-0148-x.][Negri RM, Prypsztejn HE (2001). “An Experiment on Photochromism and Kinetics for the Undergraduate Laboratory”. Journal of Chemical Education. 78 (5): 645. doi:10.1021/ed078p645.][Itoh K, Okamoto T, Wakita S, Niikura H, Murabayashi M (1991). “Thin films of peroxopolytungstic acids: applications to optical waveguide components”. Applied Organometallic Chemistry. 5 (4): 295. doi:10.1002/aoc.590050413.][Bertelson R (2002). Spiropyrans. Organic Photochromic and Thermochromic Compounds. Topics in Applied Chemistry. Vol. 5. pp. 11–83. doi:10.1007/0-306-46911-1_2. ISBN 978-0-306-45882-8.]}
            • Quinoline Yellow SS is a bright yellow dye with green shade. It is insoluble in water, but soluble in nonpolar organic solvents. Quinoline yellow is representative of a large class of quinophthalone pigments.^{[Volker Radtke “Quinophthalone Pigments” in High Performance Pigments (2nd Edition), Edited by Edwin B. Faulkner, Russell J. Schwartz, 2009 Wiley-VCH, Weinheim. doi:10.1002/9783527626915.ch19]} It is suggested that quinoline yellow exhibits excited-state intramolecular proton transfer (ESIPT) behavior and the behavior might be the cause of its decent photostability, by recent spectroscopic study.^{[Gi Rim Han et al., “Shedding new light on an old molecule: quinophthalone displays uncommon N-to-O excited state intramolecular proton transfer (ESIPT) between photobases”, Scientific Reports, 2017, 7, 3863.]}
            • As first described in 1878, the dye is prepared by the fusion of phthalic anhydride and quinaldine.
              • Quinaldine sulfate is an anaesthetic used in fish transportation.^{[Blasiola G. C. Jr. (1977). “Quinaldine sulphate, a new anaesthetic formulation for tropical marine fishes”. Journal of Fish Biology. 10 (2): 113–119(7). doi:10.1111/j.1095-8649.1977.tb04048.x.]} In some Caribbean islands it is used to facilitate the collection of tropical fish from reefs.
              • Quinaldine is used in manufacturing anti-malaria drugs, dyes and food colorants (e.g., Quinoline Yellows, pinacyanol). It is the precursor to the pH indicator Quinaldine Red.
                • Quinaldine red {QR)^{[Salem, Ibrahim A.; El-Maazawi, M. (2001). “Kinetics and Mechanism of the Homogeneous Oxidation of Quinaldine Red by Hydrogen Peroxide”. Zeitschrift für Physikalische Chemie. 215 (5): 623–6.]} is a dark green–red or black solid that does not dissolve easily in water (it is partly miscible).^{[Guesten, Hans (2006). “Note on the phototropic reaction of quinaldine red” (PDF). Retrieved April 12, 2013.]} In addition to being used as colored indicator, quinaldine red is also used as a fluorescence probe and an agent in bleaching. Quinaldine red has the ability to fluoresce. Free quinaldine red does not fluoresce in solution when it is not bound to anything, making quinaldine red only visible by fluorescence when it is bound to something. Quinaldine red can exhibit fluorescence when it is bound to nucleic acids, which then emit radiation between 580-650 nm. ^{[ Li, Wen-You; Miao, Kun; Wu, Hui-Ling; He, Xi-Wen; et al. (2003). “The Fluorescent Reaction Between Quinaldine Red and Nucleic Acids and its Application to Fluorescent Assay of DNA and RNA”. Microchimica Acta. 143: 33–7. doi:10.1007/s00604-003-0032-2. S2CID 94415153.]} The dye’s ability to bind to proteins makes it a great tag. When exposed to intensive rays such as X-rays, gamma rays, and electron beams, the dye is able to photobleach a substance. In the case of dental bleaching, a laser is the source of intensive rays. QR is dissolved in a mixture of water, ethanol, isopropyl alcohol, glycerol, and other solvents and is placed on the teeth. In the presence of oxygen, the QR and carrier particles solution uses its sensitivity to light energy to ultimately bleach teeth, making them whiter.^{[Kutsch, V. (2000). “Dental bleaching compositions, kits & methods”. Retrieved April 4, 2013.]}
              • Quinaldine or 2-methylquinoline is an organic compound with the formula CH₃C₉H₆N. It is one of the methyl derivatives of the heterocyclic compoundquinoline. It is bioactive and is used in the preparation of various dyes. It is a colorless oil but commercial samples can appear colored.^{[Gerd Collin; Hartmut Höke. “Quinoline and Isoquinoline”. Ullmann’s Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a22_465.]}
              • Quinaldine is recovered from coal tar. It can be prepared from aniline and paraldehyde via Skraup synthesis or from aniline and crotonaldehyde via Doebner-von Miller variation of the Skraup reaction.^[1]
              • Hydrogenation of quinaldine gives 2-methyltetrahydroquinoline. This reduction can be conducted enantioselectively.^{[Chen, Fei; Ding, Zi-Yuan; He, Yan-Mei; Fan, Qing-Hua (2015). “Synthesis of Optically Active 1,2,3,4-Tetrahydroquinolines via Asymmetric Hydrogenation Using Iridium-Diamine Catalyst”. Org. Synth. 92: 213–226. doi:10.15227/orgsyn.092.0213.]}
            • The compound exists as a mixture of two tautomers.^{[Horst Berneth (2008). “Methine Dyes and Pigments”. Ullmann’s Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a16_487.pub2.] [Han, G., Hwang, D., Lee, S. et al. Shedding new light on an old molecule: quinophthalone displays uncommon N-to-O excited state intramolecular proton transfer (ESIPT) between photobases. Sci Rep 7, 3863 (2017). https://doi.org/10.1038/s41598-017-04114-9}] Using other anhydrides and other quinaldine derivatives other dyes in the quinophthalone family can be prepared.
            • When sulfonated, it converts to a water-soluble derivative, Quinoline Yellow WS.
              • Quinoline Yellow WS is a mixture of organic compounds derived from the dye Quinoline Yellow SS (spirit soluble). Owing to the presence of sulfonate groups, the WS dyes are water-soluble (WS). It is a mixture of disulfonates (principally), monosulfonates and trisulfonates of 2-(2-quinolyl)indan-1,3-dione with a maximum absorption wavelength of 416 nm.^{[oint FAO/WHO Expert Committee on Food Additives (1991). Guide to specifications for general notices: general analytical techniques, identification tests, test solutions, and other reference materials (Rev. 2 [= ed. 1991] ed.). Rome: Food and Agriculture Organization of the United Nations. ISBN 9789251029916.]p. 119} Quinoline Yellow is used as a greenish yellow food additive in certain countries, designated in Europe as the E number E104.^{[ “Current EU approved additives and their E Numbers“, Food Standards Agency website, retrieved 15 Dec 2011]} In the EU and Australia, Quinoline Yellow is permitted in beverages and is used in foods, like sauces, decorations, and coatings; Quinoline Yellow is not listed as a permitted food additive in Canada or the US, where it is permitted in medicines and cosmetics and is known as D&C Yellow 10.^{[ Abbey J, et at. Colorants. pp 459-465 in Encyclopedia of Food Safety, Vol 2: Hazards and Diseases. Eds, Motarjemi Y et al. Academic Press, 2013. ISBN 9780123786135]: 461} The Codex Alimentarius does not list it.^{[ Abbey J, et at. Colorants. pp 459-465 in Encyclopedia of Food Safety, Vol 2: Hazards and Diseases. Eds, Motarjemi Y et al. Academic Press, 2013. ISBN 9780123786135]: 461}
              • Health effects
              • Quinoline Yellow WS has not been associated with any significant long-term toxicity, is not genotoxic or carcinogenic and there is no evidence of adverse effects on reproduction or development.^{[ Abbey J, et at. Colorants. pp 459-465 in Encyclopedia of Food Safety, Vol 2: Hazards and Diseases. Eds, Motarjemi Y et al. Academic Press, 2013. ISBN 9780123786135]} Food colorants in general have been the subject of much scrutiny for their effect on health.^{[Amchova, Petra; Kotolova, Hana; Ruda-Kucerova, Jana “Health safety issues of synthetic food colorants” Regulatory Toxicology and Pharmacology (2015), 73(3), 914-922.doi:10.1016/j.yrtph.2015.09.026]}
              • Possible cause of hyperactivity
              • Since the 1970s and the well-publicized advocacy of Benjamin Feingold, there has been public concern that food colorings may cause ADHD-like behavior in children.^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011]} These concerns have led the U.S. FDA and other food safety authorities to regularly review the scientific literature, and led the UK FSA to commission a study by researchers at the University of Southampton to assess the effect of a mixture of six food dyes (Tartrazine, Allura Red, Ponceau 4R, Quinoline Yellow WS, Sunset Yellow FCF and Carmoisine (dubbed the “Southampton 6”)) and sodium benzoate (a preservative) on children in the general population, who consumed them in beverages; the study published in 2007.^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011][Sarah Chapman of Chapman Technologies on behalf of Food Standards Agency in Scotland. March 2011 [Guidelines on approaches to the replacement of Tartrazine, Allura Red, Ponceau 4R, Quinoline Yellow, Sunset Yellow and Carmoisine in food and beverages]]} The study found “a possible link between the consumption of these artificial colours and a sodium benzoate preservative and increased hyperactivity” in the children;^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011][Sarah Chapman of Chapman Technologies on behalf of Food Standards Agency in Scotland. March 2011 [Guidelines on approaches to the replacement of Tartrazine, Allura Red, Ponceau 4R, Quinoline Yellow, Sunset Yellow and Carmoisine in food and beverages]]} the advisory committee to the FSA that evaluated the study also determined that because of study limitations, the results could not be extrapolated to the general population, and further testing was recommended“.^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011]}
              • The European regulatory community, with a stronger emphasis on the precautionary principle, required labelling and temporarily reduced the acceptable daily intake (ADI) for the food colorings; the UK FSA called for voluntary withdrawal of the colorings by food manufacturers.^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011][Sarah Chapman of Chapman Technologies on behalf of Food Standards Agency in Scotland. March 2011 [Guidelines on approaches to the replacement of Tartrazine, Allura Red, Ponceau 4R, Quinoline Yellow, Sunset Yellow and Carmoisine in food and beverages]]} However, in 2009 the EFSA re-evaluated the data at hand and determined that “the available scientific evidence does not substantiate a link between the color additives and behavioral effects”.^{[ FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011][EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS (November 2009). “Scientific Opinion on the re-evaluation of Quinoline Yellow (E 104) as a food additive”. EFSA Journal. 7 (11): 1329. doi:10.2903/j.efsa.2009.1329.]} On the basis of other evidence the EFSA also reduced the acceptable daily intake (ADI) from 10 to 0.5 mg/kg.^{[EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS (November 2009). “Scientific Opinion on the re-evaluation of Quinoline Yellow (E 104) as a food additive”. EFSA Journal. 7 (11): 1329. doi:10.2903/j.efsa.2009.1329.]}
              • The US FDA did not make changes following the publication of the Southampton study, but following a citizen petition filed by the Center for Science in the Public Interest in 2008, requesting the FDA ban several food additives, the FDA commenced a review of the available evidence, and still made no changes.^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011]}
              • No evidence supports broad claims that food coloring causes food intolerance and ADHD-like behavior in children.^{[Tomaska LD and Brooke-Taylor, S. Food Additives – General pp 449-454 in Encyclopedia of Food Safety, Vol 2: Hazards and Diseases. Eds, Motarjemi Y et al. Academic Press, 2013. ISBN 9780123786135]: 452} It is possible that certain food coloring may act as a trigger in those who are genetically predisposed, but the evidence is weak.^{[FDA. Background Document for the Food Advisory Committee: Certified Color Additives in Food and Possible Association with Attention Deficit Hyperactivity Disorder in Children: March 30-31, 2011][ Millichap JG, Yee MM (February 2012). “The diet factor in attention-deficit/hyperactivity disorder”. Pediatrics. 129 (2): 330–337. doi:10.1542/peds.2011-2199. PMID 22232312. S2CID 14925322.]}
            • Quinoline Yellow SS is used in spirit lacquers, polystyrene, polycarbonates, polyamides, acrylic resins, and to color hydrocarbon solvents. It is also used in externally applied drugs and cosmetics. Quinoline Yellow SS is used in some yellow colored smoke formulations.
            • It may cause contact dermatitis. It has the appearance of a yellow powder with a melting point of 240 °C (464 °F).
          • Apocyanines
            • where two quaternary nitrogens are joined by a polymethine chain.^{[Ernst LA, Gupta RK, Mujumdar RB, Waggoner AS (Jan 1989). “Cyanine dye labeling reagents for sulfhydryl groups”. Cytometry. 10 (1): 3–10. doi:10.1002/cyto.990100103. PMID 2917472.]} Both nitrogens may each be independently part of a heteroaromatic moiety, such as pyrrole, imidazole, thiazole, pyridine, quinoline, indole, benzothiazole, etc.
            • History and use in industry
              • Cyanines were first synthesized over a century ago. They were originally used, and still are, to increase the sensitivity range of photographic emulsions, i.e., to increase the range of wavelengths which will form an image on the film, making the film panchromatic.^{[Berneth, Horst (2008). “Methine Dyes and Pigments”. Ullmann’s Encyclopedia of Industrial Chemistry. Weinheim: Wiley-VCH. doi:10.1002/14356007.a16_487.pub2.]} Cyanines are also used in CD-R and DVD-R media. The ones used are mostly green or light blue colour, and are chemically unstable. For that reason, unstabilized cyanine discs are unsuitable for archival CD and DVD use. Recent cyanine discs contain stabilizers, typically a metal atom bonded to the cyanine molecule,^{[“Archival Lifespan of TDK CD-R”. cdrom2go.com. US Digital Media. Retrieved 3 April 2022.]} that slow the deterioration significantly. These discs are often rated with an archival life of 75 years or more. The other dyes used in CD-Rs are phthalocyanine and azo.
              • Use in biotechnology
                • For applications to biotechnology, special cyanine dyes are synthesized from 2, 3, 5 or 7-methine structures with reactive groups on either one or both of the nitrogen ends so that they can be chemically linked to either nucleic acids or protein molecules. Labeling is done for visualization and quantification purposes. Biological applications include comparative genomic hybridization and gene chips, which are used in transcriptomics, and various studies in proteomics such as RNA localization,^{[Blower MD, Feric E, Weis K, Heald R (Dec 2007). “Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules”. The Journal of Cell Biology. 179 (7): 1365–73. doi:10.1083/jcb.200705163. PMC 2373496. PMID 18166649.]} molecular interaction studies by fluorescence resonance energy transfer (FRET) and fluorescent immunoassays.
                • Cyanine dyes are available with different modifications such as methyl, ethyl or butyl substituents, carboxyl, acetylmethoxy, and sulfo groups which alter their hydrophilicity.^{[CYanine dyes]}
              • Common cyanine dyes and their uses
                • Because they yield brighter and more stable fluorescence, cyanines can advantageously replace conventional dyes such as fluorescein and rhodamines.
                • Cy3 and Cy5 are the most popular, used typically combined for 2 colors detection.
                • Cy3 fluoresces greenish yellow (~550 nm excitation, ~570 nm emission), while Cy5 is fluorescent in the far-red region (~650 excitation, 670 nm emission).^{[Jackson ImmunoResearch. “Cyanine Dyes (Cy2, Cy3, and Cy5)”. Retrieved 2008-10-31.]} Cy3 can be detected by various fluorometers, imagers, and microscopes with standard filters for Tetramethylrhodamine (TRITC). Due to its high molar extinction coefficient, this dye is also easily detected by naked eye on electrophoresis gels, and in solution. Cy5 became a popular replacement for far red fluorescent dyes because of its high extinction coefficient (as small as 1 nanomol can be detected in gel electrophoresis by naked eye) and its fluorophore emission maximum in the red region, where many CCD detectors have maximum sensitivity and biological objects give low background interference.
                • The scanners actually use diverse laser emission wavelengths (typically 532 nm and 635 nm) and filter wavelengths (550-600 nm and 655-695 nm) to avoid background contamination. They are thus able to easily distinguish colors from Cy3 and from Cy5, and also able to quantify the amount of Cy3 and Cy5 labeling in one sample (multiparametric detection).
                • Other cyanine dyes are useful:
                  • Cy3.5 can replace sulfoRhodamine 101.
                  • Cy5.5 is a near-infrared (IR) fluorescence-emitting dye (excitation/emission maximum 678/694 nm).
                  • Cy7 is a near-IR fluor that is invisible to the naked eye (excitation/emission maximum 750/776 nm). It is used in in vivo imaging applications, as well as the Cy7.5 dye.
                  • Sulfo–Cyanine dyes bear one or two sulfo groups, rendering the Cy dye water-soluble, but tri- and quadri-sulfonated forms are available for even higher water solubility.^{[CYanine dyes]} PEGylation is another modification that confers hydrophilicity, not only to the dye but also to the labeled conjugate.
              • Nomenclature and structure
                • The Cy3 and Cy5 nomenclature was first proposed by Ernst, et al.^{[Ernst LA, Gupta RK, Mujumdar RB, Waggoner AS (Jan 1989). “Cyanine dye labeling reagents for sulfhydryl groups”. Cytometry. 10 (1): 3–10. doi:10.1002/cyto.990100103. PMID 2917472.]} in 1989, and is non-standard since it gives no hint of their chemical structures. In the original paper the number designated the count of the methines (as shown), and the side chains were unspecified. Due to this ambiguity various structures are designated Cy3 and Cy5 in the literature. The R groups do not have to be identical. In the dyes as used they are short aliphatic chains one or both of which ends in a highly reactive moiety such as N-hydroxysuccinimide or maleimide.
              • Alternatives
                • Many analogs of standard Cy 2 / 3 / 3.5 / 5 / 5.5 / 7 / 7.5 dyes were developed, using diverse modification: Alexa Fluor dyes, Dylight, FluoProbes dyes, Sulfo Cy dyes,^{[“Cyandye, LLC”. Archived from the original on 2018-10-03. Retrieved 2013-11-01.]} Seta dyes,^{[ SETA BioMedicals]} IRIS dyes from Cyanine Technologies ^{[“Iris Dyes | Cyanine Technologies”. Archived from the original on 2015-01-26. Retrieved 2015-01-26]} and others can be used interchangeably with Cy dyes in most biochemical applications, with claimed improvements in solubility, fluorescence, or photostability.^{[ FluoProbes488 comparison to FITC, Cyanine2][FluoProbes547H comparison in Confocal Microscopy]}
                • While patent protection for the standard Cy series of dyes has lapsed, the trademarked Cy naming remains in place. Consequently, dyes that are identical to Cy dyes, but called different names, are now sold.
              • Applications
                • Cyanine dyes are used to label proteins, antibodies, peptides, nucleic acid probes, and any kind of other biomolecules to be used in a variety of fluorescence detection techniques: Flow cytometry, Microscopy (mainly Visible range, but also UV, IR), Microplate assays, Microarrays, as well as “light-up Probes,” and in vivo imaging.^{[Armitage, Bruce A. (27 January 2005). “Cyanine Dye–DNA Interactions: Intercalation, Groove Binding, and Aggregation”. DNA Binders and Related Subjects. Topics in Current Chemistry. Vol. 253. pp. 55–76. doi:10.1007/b100442. ISBN 978-3-540-22835-6.]}
              • Nucleic acid labeling
                • In microarray experiments DNA or RNA is labeled with either Cy3 or Cy5 that has been synthesized to carry an N-hydroxysuccinimidyl ester (NHS-ester) reactive group. Since NHS-esters react readily only with aliphatic amine groups, which nucleic acids lack, nucleotides have to be modified with aminoallyl groups. This is done through incorporating aminoallyl-modified nucleotides during synthesis reactions. A good ratio is a label every 60 bases such that the labels are not too close to each other, which would result in quenching effects.
              • Protein labeling
                • For protein labeling, Cy3 and Cy5 dyes sometimes bear a succinimidyl group to react with amines, or a maleimide group to react with a sulfhydryl group of cysteine residues.
                • Cy5 is sensitive to its electronic environment. Changes in the conformation of the protein it is attached to will produce either enhancement or quenching of the emission. The rate of this change can be measured to determine enzyme kinetic parameters. The dyes can be used for similar purposes in FRET experiments.
                • Cy3 and Cy5 are used in proteomics experiments so that samples from two sources can be mixed and run together through the separation process.^[19][20] This eliminates variations due to differing experimental conditions that are inevitable if the samples were run separately. These variations make it extremely difficult, if not impossible, to use computers to automate the acquisition of the data after the separation is complete. Using these dyes makes the automation trivial.
              • Etymology
                • The word cyanin is from the English word “cyan”, which conventionally means a shade of blue-green (close to “aqua”) and is derived from the Greek κυάνεος/κυανοῦς kyaneos/kyanous which means a somewhat different color: “dark blue”.
      • Bilirubin
        • Bilirubin (BR) (from the Latin for “red bile”) is a red-orange compound that occurs in the normal catabolic pathway that breaks down heme in vertebrates. This catabolism is a necessary process in the body’s clearance of waste products that arise from the destruction of aged or abnormal red blood cells.^{[ Braunstein E (3 May 2019). “Overview of Hemolytic Anemia – Hematology and Oncology”. Merck Manuals Professional Edition (in Latin). Retrieved 5 May 2019.]} In the first step of bilirubin synthesis, the heme molecule is stripped from the hemoglobin molecule. Heme then passes through various processes of porphyrin catabolism, which varies according to the region of the body in which the breakdown occurs. For example, the molecules excreted in the urine differ from those in the feces.^{[ “Bilirubin blood test”, U.S. National Library of Medicine.]} The production of biliverdin from heme is the first major step in the catabolic pathway, after which the enzyme biliverdin reductase performs the second step, producing bilirubin from biliverdin.^{[Boron W, Boulpaep E. Medical Physiology: a cellular and molecular approach, 2005. 984–986. Elsevier Saunders, United States. ISBN 1-4160-2328-3][Mosqueda L, Burnight K, Liao S (August 2005). “The life cycle of bruises in older adults”. Journal of the American Geriatrics Society. 53 (8): 1339–43. doi:10.1111/j.1532-5415.2005.53406.x. PMID 16078959. S2CID 12394659.]}
        • Ultimately, bilirubin is broken down within the body, and its metabolites excreted through bile and urine; elevated levels may indicate certain diseases.^{[Smith ME, Morton DG (2010). “LIVER AND BILIARY SYSTEM”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]} It is responsible for the yellow color of healing bruises and the yellow discoloration in jaundice. Its breakdown products, such as stercobilin, cause the brown color of feces. A different breakdown product, urobilin, is the main component of the straw-yellow color in urine.^{[citation needed]}
        • Although bilirubin is usually found in animals rather than plants, at least one plant species, Strelitzia nicolai, is known to contain the pigment.^{[Pirone C, Quirke JM, Priestap HA, Lee DW (March 2009). “Animal pigment bilirubin discovered in plants”. Journal of the American Chemical Society. 131 (8): 2830. doi:10.1021/ja809065g. PMC 2880647. PMID 19206232.]}
          • Strelitzia nicolai, commonly known as the wild banana or giant white bird of paradise, is a species of banana-like plants with erect woody stems reaching a height of 7–8 m (23–26 ft), and the clumps formed can spread as far as 3.5 m (11 ft).
          • The 1.8 m (5 ft 11 in)-long leaves are grey-green and arranged like a fan at the top of the stems, similar to Ravenala madagascariensis. The inflorescence is composed of a dark blue bract, white sepals and a bluish-purple “tongue”. The entire flower can be as much as 18 cm (7.1 in) high by 45 cm (18 in) long, and is typically held just above the point where the leaf fan emerges from the stem. Flowers are followed by triangular seed capsules.^{[Gartenbau-Gesellschaft., Deutsche; Petersburg., Kaiserliche Russischer Gartenbau-Verein in St.; Petersburg., Russischer Gartenbau-Verein in St.; Staaten., Verein zur Beförderung des Gartenbaues in den Königlich Preussischen (1858). “Gartenflora”. Jahrg.7 (1858).][Gibbs Russell, G. E., W. G. M. Welman, E. Retief, K. L. Immelman, G. Germishuizen, B. J. Pienaar, M. Van Wyk & A. Nicholas. 1987. List of species of southern African plants. Memoirs of the Botanical Survey of South Africa 2(1–2): 1–152(pt. 1), 1–270(pt. 2), Strelitzia nicolai]}
          • Strelitzia nicolai is among the few plants which have been verified to contain the pigment bilirubin, which is usually found in animals.^{[ Pirone, Cary; Quirke, J. Martin E.; Priestap, Horacio A.; Lee, David W. (2009). “Animal Pigment Bilirubin Discovered in Plants”. Journal of the American Chemical Society. 131 (8): 2830. doi:10.1021/ja809065g. PMC 2880647. PMID 19206232.]}
          • Strelitzia nicolai is one of three larger Strelitzia species, the other two being tree-like S. caudata and S. alba. S. nicolai is restricted to evergreen coastal forest and thicket of eastern South Africa from the Gonubie northwards to southern Mozambique. It is also considered native to Mozambique, Botswana and Zimbabwe, and is reportedly naturalized in eastern Mexico (State of Veracruz).^{[ “World Checklist of Selected Plant Families: Royal Botanic Gardens, Kew”. apps.kew.org. Retrieved 2017-08-01.]}
        • Structure
          • Bilirubin consists of an open-chain tetrapyrrole. It is formed by oxidative cleavage of a porphyrin in heme, which affords biliverdin. Biliverdin is reduced to bilirubin. After conjugation with glucuronic acid, bilirubin is excreted.^{[citation needed]}
          • Bilirubin is structurally similar to the pigment phycobilin used by certain algae to capture light energy, and to the pigment phytochrome used by plants to sense light. All of these contain an open chain of four pyrrolic rings.^{[citation needed]}
          • Like these other pigments, some of the double-bonds in bilirubin isomerize when exposed to light. This isomerization is relevant to the phototherapy of jaundiced newborns: the E,Z-isomers of bilirubin formed upon light exposure are more soluble than the unilluminated Z,Z-isomer, as the possibility of intramolecular hydrogen bonding is removed.^{[McDonagh AF, Palma LA, Lightner DA (April 1980). “Blue light and bilirubin excretion”. Science. 208 (4440): 145–51. Bibcode:1980Sci…208..145M. doi:10.1126/science.7361112. PMID 7361112.]} Increased solubility allows the excretion of unconjugated bilirubin in bile.
          • Some textbooks and research articles show the incorrect geometric isomer of bilirubin.^{[“Bilirubin’s Chemical Formula”. Archived from the original on 4 May 2011. Retrieved 14 August 2007.]} The naturally occurring isomer is the Z,Z-isomer.
        • Function
          • Bilirubin is created by the activity of biliverdin reductase on biliverdin, a green tetrapyrrolic bile pigment that is also a product of heme catabolism. Bilirubin, when oxidized, reverts to become biliverdin once again. This cycle, in addition to the demonstration of the potent antioxidant activity of bilirubin,^{[Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN (February 1987). “Bilirubin is an antioxidant of possible physiological importance”. Science. 235 (4792): 1043–6. Bibcode:1987Sci…235.1043S. doi:10.1126/science.3029864. PMID 3029864.]} has led to the hypothesis that bilirubin’s main physiologic role is as a cellular antioxidant.^{[Baranano DE, Rao M, Ferris CD, Snyder SH (December 2002). “Biliverdin reductase: a major physiologic cytoprotectant”. Proceedings of the National Academy of Sciences of the United States of America. 99 (25): 16093–8. Bibcode:2002PNAS…9916093B. doi:10.1073/pnas.252626999. JSTOR 3073913. PMC 138570. PMID 12456881.][Sedlak TW, Saleh M, Higginson DS, Paul BD, Juluri KR, Snyder SH (March 2009). “Bilirubin and glutathione have complementary antioxidant and cytoprotective roles”. Proceedings of the National Academy of Sciences of the United States of America. 106 (13): 5171–6. Bibcode:2009PNAS..106.5171S. doi:10.1073/pnas.0813132106. JSTOR 40455167. PMC 2664041. PMID 19286972.]} Consistent with this, animal studies suggest that eliminating bilirubin results in endogenous oxidative stress.^{[Chen W, Maghzal GJ, Ayer A, Suarna C, Dunn LL, Stocker R (February 2018). “Absence of the biliverdin reductase-a gene is associated with increased endogenous oxidative stress”. Free Radical Biology & Medicine. 115: 156–165. doi:10.1016/j.freeradbiomed.2017.11.020. PMID 29195835. S2CID 25089098.]} Bilirubin’s antioxidant activity may be particularly important in the brain, where it prevents excitotoxicity and neuronal death by scavenging superoxide during N-methyl-D-aspartic acid neurotransmission.^{[Vasavda C, Kothari R, Malla AP, Tokhunts R, Lin A, Ji M, et al. (October 2019). “Bilirubin Links Heme Metabolism to Neuroprotection by Scavenging Superoxide”. Cell Chemical Biology. 26 (10): 1450–1460.e7.]}
        • Metabolism
          • Total bilirubin = direct bilirubin + indirect bilirubin^[16]
          • 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^{[Gwaltney-Brant SM (2016). “Nutraceuticals in Hepatic Diseases”. Nutraceuticals. Elsevier. pp. 87–99. doi:10.1016/b978-0-12-802147-7.00007-3. ISBN 978-0-12-802147-7. S2CID 78381597.]}
          • 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.^{[“Unconjugated Hyperbilirubinemia: Practice Essentials, Background, Pathophysiology”. Medscape Reference. 4 March 2019. Retrieved 6 May 2019.][“Bilirubin: Reference Range, Interpretation, Collection and Panels”. Medscape Reference. 1 February 2019. Retrieved 6 May 2019.]}
          • Direct
            • Direct bilirubin = Conjugated bilirubin + delta bilirubin^{[Tietze KJ (2012). “Review of Laboratory and Diagnostic Tests”. Clinical Skills for Pharmacists. Elsevier. pp. 86–122. doi:10.1016/b978-0-323-07738-5.10005-5. ISBN 978-0-323-07738-5.]}
            • 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 colon.^{[Cheifetz AS (2010). Oxford American Handbook of Gastroenterology and Hepatology. Oxford: Oxford University Press, USA. p. 165. ISBN 978-0199830121.]}
              • 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.^{[Kuntz, Erwin (2008). Hepatology: Textbook and Atlas. Germany: Springer. p. 38. ISBN 978-3-540-76838-8.]}
              • Conjugated bilirubin’s half-life is shorter than delta bilirubin.^{[Sullivan KM, Gourley GR (2011). “Jaundice”. Pediatric Gastrointestinal and Liver Disease. Elsevier. pp. 176–186.e3. doi:10.1016/b978-1-4377-0774-8.10017-x. ISBN 978-1-4377-0774-8.]}
            • Delta bilirubin
              • Although the terms direct and indirect bilirubin are used equivalently with conjugated and unconjugated bilirubin, this is not quantitatively correct, because the direct fraction includes both conjugated bilirubin and δ bilirubin.^{[citation needed]}
              • Delta bilirubin is albumin-bound conjugated bilirubin.^{[Tietze KJ (2012). “Review of Laboratory and Diagnostic Tests”. Clinical Skills for Pharmacists. Elsevier. pp. 86–122. doi:10.1016/b978-0-323-07738-5.10005-5. ISBN 978-0-323-07738-5.]} 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.^{[ Moyer KD, Balistreri WF (2011). “Liver Disease Associated with Systemic Disorders”. In Kliegman RM, Stanton BF, St Geme JW, Schor NF, Behrman RE (eds.). Nelson Textbook of Pediatrics. Saunders. p. 1405. ISBN 978-1-4377-0755-7.]} 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).
              • δ bilirubin = total bilirubin – (unconjugated bilirubin + conjugated bilirubin)^{[Tietze KJ (2012). “Review of Laboratory and Diagnostic Tests”. Clinical Skills for Pharmacists. Elsevier. pp. 86–122. doi:10.1016/b978-0-323-07738-5.10005-5. ISBN 978-0-323-07738-5.]}
            • Half-life
              • The half-life of delta bilirubin is equivalent to that of albumin since the former is bound to the latter, yields 2–3 weeks.^{[ Kalakonda A, John S (2019). “Physiology, Bilirubin article-18281”. StatPearls. Treasure Island (FL): StatPearls Publishing. PMID 29261920. Retrieved 22 December 2019. 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).][“Unconjugated Hyperbilirubinemia: Practice Essentials, Background, Pathophysiology”. Medscape Reference. 4 March 2019. Retrieved 6 May 2019.]}
              • A free-of-bound bilirubin has a half-life of 2 to 4 hours.^{[ Kalakonda A, John S (2019). “Physiology, Bilirubin article-18281”. StatPearls. Treasure Island (FL): StatPearls Publishing. PMID 29261920. Retrieved 22 December 2019. 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).]}
              • Further information: Bilirubin glucuronide
          • Urine
            • Under normal circumstances, only a very small amount, if any, of urobilinogen, is excreted in the urine. If the liver’s function is impaired or when biliary drainage is blocked, some of the conjugated bilirubin leaks out of the hepatocytes and appears in the urine, turning it dark amber. However, in disorders involving hemolytic anemia, an increased number of red blood cells are broken down, causing an increase in the amount of unconjugated bilirubin in the blood. Because the unconjugated bilirubin is not water-soluble, one will not see an increase in bilirubin in the urine. Because there is no problem with the liver or bile systems, this excess unconjugated bilirubin will go through all of the normal processing mechanisms that occur (e.g., conjugation, excretion in bile, metabolism to urobilinogen, reabsorption) and will show up as an increase of urobilinogen in the urine. This difference between increased urine bilirubin and increased urine urobilinogen helps to distinguish between various disorders in those systems.^{[ Roxe, D. M.; Walker, H. K.; Hall, W. D.; Hurst, J. W. (1990). “Urinalysis”. Clinical Methods: The History, Physical, and Laboratory Examinations. Butterworths. ISBN 9780409900774. PMID 21250145.]}
          • Toxicity
            • Main article: Kernicterus
            • Unbound bilirubin (Bf) levels can be used to predict the risk of neurodevelopmental handicaps within infants.^{[Hegyi, T.; Chefitz, D.; Weller, A.; Huber, A; Carayannopoulos, M.; Kleinfeld, A. (2020). “Unbound bilirubin measurements in term and late-preterm infants”. Journal of Maternal-Fetal & Neonatal Medicine. 35 (8): 1532–1538. doi:10.1080/14767058.2020.1761318. PMC 7609464. PMID 32366186.]} Unconjugated hyperbilirubinemia in a newborn can lead to accumulation of bilirubin in certain brain regions (particularly the basal nuclei) with consequent irreversible damage to these areas manifesting as various neurological deficits, seizures, abnormal reflexes and eye movements. This type of neurological injury is known as kernicterus. The spectrum of clinical effect is called bilirubin encephalopathy. The neurotoxicity of neonatal hyperbilirubinemia manifests because the blood–brain barrier has yet to develop fully,^{[dubious – discuss]} and bilirubin can freely pass into the brain interstitium, whereas more developed individuals with increased bilirubin in the blood are protected. Aside from specific chronic medical conditions that may lead to hyperbilirubinemia, neonates in general are at increased risk since they lack the intestinal bacteria that facilitate the breakdown and excretion of conjugated bilirubin in the feces (this is largely why the feces of a neonate are paler than those of an adult). Instead the conjugated bilirubin is converted back into the unconjugated form by the enzyme β-glucuronidase (in the gut, this enzyme is located in the brush border of the lining intestinal cells) and a large proportion is reabsorbed through the enterohepatic circulation. In addition, recent studies point towards high total bilirubin levels as a cause for gallstones regardless of gender or age.^{[Zeng, D.; Wu, H.; Huang, Q.; Zeng, A.; Yu, Z.; Zhong, Z. (2021). “High levels of serum triglyceride, low-density lipoprotein cholesterol, total bile acid, and total bilirubin are risk factors for gallstones”. Clinical Laboratory. 67 (8): 1905–1913. doi:10.7754/Clin.Lab.2021.201228. PMID 34383399. S2CID 234775572. Retrieved 11 November 2021 – via PubMed.]}
          • Health benefits
            • In the absence of liver disease, high levels of total bilirubin confers various health benefits.^{[Sedlak TW, Snyder SH (June 2004). “Bilirubin benefits: cellular protection by a biliverdin reductase antioxidant cycle”. Pediatrics. 113 (6): 1776–82. doi:10.1542/peds.113.6.1776. PMID 15173506.]} Studies have also revealed that levels of serum bilirubin (SBR)^{[“Neonatal Jaundice”. Slhd.nsw.gov.au. 24 August 2009. Retrieved 16 March 2022.]} are inversely related to risk of certain heart diseases.^{[Novotný L, Vítek L (May 2003). “Inverse relationship between serum bilirubin and atherosclerosis in men: a meta-analysis of published studies”. Experimental Biology and Medicine. 228 (5): 568–71. doi:10.1177/15353702-0322805-29. PMID 12709588. S2CID 43486067.][Schwertner HA, Vítek L (May 2008). “Gilbert syndrome, UGT1A1*28 allele, and cardiovascular disease risk: possible protective effects and therapeutic applications of bilirubin”. Atherosclerosis. 198 (1): 1–11. doi:10.1016/j.atherosclerosis.2008.01.001. PMID 18343383.]} While the poor solubility and potential toxicity of bilirubin limit its potential medicinal applications, current research is being done on whether bilirubin encapsulated silk fibrin nanoparticles can alleviate symptoms of disorders such as acute pancreatitis.^{[Yao, Q.; Jiang, X.; Zhai, Yuan-Yuan; Luo, Lan-Zi; Xu, He-Lin; Xiao, J.; Kou, L.; zhao, Ying-Zheng (2020). “Protective effects and mechanisms of bilirubin nanomedicine against acute pancreatitis”. Journal of Controlled Release. 332: 312–325. doi:10.1016/j.jconrel.2020.03.034. PMID 32243974. S2CID 214786812. Retrieved 11 November 2021 – via Elsevier Science Direct.]} In addition to this, there has been recent discoveries linking bilirubin and its ε-polylysine-bilirubin conjugate (PLL-BR), to more efficient insulin medication. It seems that bilirubin exhibits protective properties during the islet transplantation process when drugs are delivered throughout the bloodstream.^{[Zhao, Ying-Zheng; Huang, Zhi-Wei; Zhai, Yuan-Yuan; Shi, Yannan; Du, Chu-Chu; Zhai, Jiaoyuan; Xu, He-Lin; Xiao, Jian; Kou, Longfa; Yao, Qing (2021). “Polylysine-bilirubin conjugates maintain functional islets and promote M2 macrophage polarization”. Acta Biomaterialia. 122: 172–185. doi:10.1016/j.actbio.2020.12.047. PMID 33387663. S2CID 230281925. Retrieved 11 November 2021 – via Elsevier Science Direct.]}
          • 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. For adults, blood is typically collected by needle from a vein in the arm. 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 an instrument placed on the skin (transcutaneous bilirubin meter)^{[citation needed]}
            • Bilirubin (in blood) is found in two forms:
              • “Conjugated bilirubin”
              • “Unconjugated 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]}
            • 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.
            • Indirect bilirubin is fat-soluble and direct bilirubin is water-soluble.^{[“Bilirubin: The Test | Bilirubin Test: Total bilirubin; TBIL; Neonatal bilirubin; Direct bilirubin; Conjugated bilirubin; Indirect bilirubin; Unconjugated bilirubin | Lab Tests Online”. labtestsonline.org. Retrieved 14 June 2017.]}
          • Measurement methods
            • Originally, the Van den Bergh reaction was used for a qualitative estimate of bilirubin.
            • This test is performed routinely in most medical laboratories and can be measured by a variety of methods.^{[Watson D, Rogers JA (May 1961). “A study of six representative methods of plasma bilirubin analysis”. Journal of Clinical Pathology. 14 (3): 271–8. doi:10.1136/jcp.14.3.271. PMC 480210. PMID 13783422.]}
            • 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.^{[Rolinski B, Küster H, Ugele B, Gruber R, Horn K (October 2001). “Total bilirubin measurement by photometry on a blood gas analyzer: potential for use in neonatal testing at the point of care”. Clinical Chemistry. 47 (10): 1845–7. doi:10.1093/clinchem/47.10.1845. PMID 11568098.]}
          • Blood levels
            • The bilirubin level found in the body reflects the balance between production and excretion. Blood test results are advised to always be interpreted using the reference range provided by the laboratory that performed the test. 
          • Hyperbilirubinemia
            • Hyperbilirubinemia is a higher-than-normal level of bilirubin in the blood.
            • Mild rises in bilirubin may be caused by:
            • Hemolysis or increased breakdown of red blood cells
            • Gilbert’s syndrome – a genetic disorder of bilirubin metabolism that can result in mild jaundice, found in about 5% of the population
            • Rotor syndrome: non-itching jaundice, with rise of bilirubin in the patient’s serum, mainly of the conjugated type
            • Moderate^{[clarification needed]} rise in bilirubin may be caused by:
            • Pharmaceutical drugs (especially antipsychotic, some sex hormones, and a wide range of other drugs)
              • Sulfonamides are contraindicated in infants less than 2 months old (exception when used with pyrimethamine in treating toxoplasmosis) as they increase unconjugated bilirubin leading to kernicterus.^{[“Sulfonamides: Bacteria and Antibacterial Drugs: Merck Manual Professional”.[permanent dead link]]}
              • Drugs such as protease inhibitors like Indinavir can also cause disorders of bilirubin metabolism by competitively inhibiting the UGT1A1 enzyme.^{[ Ramakrishnan, N.; Bittar, K.; Jialal, I. (8 March 2019). “Impaired Bilirubin Conjugation”. NCBI Bookshelf. PMID 29494090. Retrieved 3 May 2019.]}
            • Hepatitis (levels may be moderate or high)
            • Chemotherapy
            • Biliary stricture (benign or malignant)
            • Very high^{[clarification needed]} levels of bilirubin may be caused by:
            • Neonatal hyperbilirubinemia, where the newborn’s liver is not able to properly process the bilirubin causing jaundice
            • Unusually large bile duct obstruction, e.g. stone in common bile duct, tumour obstructing common bile duct etc.
            • Severe liver failure with cirrhosis (e.g. primary biliary cirrhosis)
            • Crigler–Najjar syndrome
            • Dubin–Johnson syndrome
            • Choledocholithiasis (chronic or acute).
            • Cirrhosis may cause normal, moderately high or high levels of bilirubin, depending on exact features of the cirrhosis.
            • To further elucidate the causes of jaundice or increased bilirubin, it is usually simpler to look at other liver function tests (especially the enzymes alanine transaminase, aspartate transaminase, gamma-glutamyl transpeptidase, alkaline phosphatase), blood film examination (hemolysis, etc.) or evidence of infective hepatitis (e.g., hepatitis A, B, C, delta, E, etc.).
          • Jaundice
            • Main article: Jaundice
            • Hemoglobin acts to transport oxygen which the body receives to all body tissue via blood vessels. Over time, when red blood cells need to be replenished, the hemoglobin is broken down in the spleen; it breaks down into two parts: heme group consisting of iron and bile and protein fraction. While protein and iron are utilized to renew red blood cells, pigments that make up the red color in blood are deposited into the bile to form bilirubin.^{[Point WW (April 1958). “Jaundice”. The American Journal of Nursing. 58 (4): 556–7. PMID 13508735.]} Jaundice leads to raised bilirubin levels that in turn negatively remove elastin-rich tissues.^{[Greenberg DA (December 2002). “The jaundice of the cell”. Proceedings of the National Academy of Sciences of the United States of America. 99 (25): 15837–9. Bibcode:2002PNAS…9915837G. doi:10.1073/pnas.012685199. PMC 138521. PMID 12461187. S2CID 30298986.]} Jaundice may be noticeable in the sclera of the eyes at levels of about 2 to 3 mg/dl (34 to 51 μmol/L),^{[Merck Manual Jaundice Last full review/revision July 2009 by Steven K. Herrine]} and in the skin at higher levels.^{[For conversion, 1 mg/dl = 17.1 μmol/L.]}
            • Jaundice is classified, depending upon whether the bilirubin is free or conjugated to glucuronic acid, into conjugated jaundice or unconjugated jaundice.^{[citation needed]}
          • Urine tests
            • Urine bilirubin may also be clinically significant.MedlinePlus Encyclopedia: Bilirubin – urine</ref> Bilirubin is not normally detectable in the urine of healthy people. If the blood level of conjugated bilirubin becomes elevated, e.g. due to liver disease, excess conjugated bilirubin is excreted in the urine, indicating a pathological process.^{[“Urinalysis: three types of examinations”. Lab Tests Online (USA). Retrieved 16 August 2013.]} Unconjugated bilirubin is not water-soluble and so is not excreted in the urine. Testing urine for both bilirubin and urobilinogen can help differentiate obstructive liver disease from other causes of jaundice.^{[Roxe, D. M.; Walker, H. K.; Hall, W. D.; Hurst, J. W. (1990). “Urinalysis”. Clinical Methods: The History, Physical, and Laboratory Examinations. Butterworths. ISBN 9780409900774. PMID 21250145.]}
          • History
            • In ancient history, Hippocrates discussed bile pigments in two of the four humours in the context of a relationship between yellow and black biles.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} 
            • Hippocrates visited Democritus in Abdera who was regarded as the expert in melancholy “black bile”.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]}
            • Relevant documentation emerged in 1827 when M. Louis Jacques Thénard examined the biliary tract of an elephant that had died at a Paris zoo. He observed dilated bile ducts were full of yellow magma, which he isolated and found to be insoluble in water. Treating the yellow pigment with hydrochloric acid produced a strong green color. Thenard suspected the green pigment was caused by impurities derived from mucus of bile.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]}
            • Leopold Gmelin experimented with nitric acid in 1826 to establish the redox behavior in change from bilirubin to biliverdin, although the nomenclature did not exist at the time.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} 
              • German chemist and professor at the University of Heidelberg He worked on the red prussiate and created Gmelin’s test, and wrote his Handbook of Chemistry, which over successive editions became a standard reference work still in use.
              • son of the physician, botanist and chemist Johann Friedrich Gmelin 
              • Focus of his research was the Black pigment of oxen and calves eyes, outcome of this work was also the subject of Gmelins dissertation. 
              • a close cooperation with Friedrich Tiedemann evolved with time. The two published “The digestion after tests” in 1826 and established the basis of the physiological chemistry. In the field of digestive chemistry Gmelin later discovered more components of bile and introduced Gmelin’s test. When Friedrich Wöhler worked on complex cyanogen compounds in 1822, Gmelin assisted him and discovered the Red prussiate.
              • From 1833 to 1838 Gmelin owned a paper mill in the north of Heidelberg situated Schriesheim, he had taken it over in the hope of profit. However, the work in the mill showed to be very time- and money-consuming and at the expense of his academic activity.
              • In 1817 the first volume of Gmelin’s Handbook of Chemistry was published. By 1843 it had grown in the fourth edition to 9 volumes. In this edition Gmelin included atomic theory and devoted much more space to the increasingly important organic chemistry. The Handbuch was published in print until the 8th edition in 1990, with an online database, which is less complete and less up-to-date than the print edition.^{[“LibGuides: Chemistry: Gmelin Handbook”. University of Texas. 29 July 2022.]} 
              • The terms ester and ketone (from German Aketon, meaning acetone) were introduced by Gmelin. Until his death Gmelin worked on the fifth edition of the handbook, which had become a valuable source of chemical information and documentation.
              • He also established the basis of an unambiguous classification of inorganic substances, later named the Gmelin system.
              • At the age of 60 Gmelin suffered a first stroke, and another in August 1850. In both strokes the right half of his body was affected; he was able to recover from the paralysis, but remained debilitated. In the spring of 1851 Gmelin applied for retirement, which was granted a few months later. In the two following years he suffered increasingly from the effects of a brain illness, at nearly 65 years Leopold Gmelin died in Heidelberg on 13 April 1853
              • In his works Leopold Gmelin dealt with physiology, mineralogy and chemistry. His experimental work was marked by his very thorough and comprehensive way of working; also some writing talent is attributed to him.
              • Gmelin’s first physiological work was his dissertation on the black pigment of oxen’s and calves’ eyes, whose coloring principle he tried to fathom. Despite the simplest chemical means he could describe the properties of the pigment and recognized the carbon rightly as the cause of staining. Gmelin’s most important physiological work was the 1826 released digestion by experiments, which he made together with Friedrich Tiedemann. The work, which also described many new working techniques, contained groundbreaking insights into the gastric juice, in which they found hydrochloric acid, and bile, in which Gmelin and Tiedemann among others discovered cholesterol and taurine. Introduced by Gmelin, Gmelin’s test enabled the detection of bile constituents in the urine of people suffering from jaundice. Furthermore, Gmelin and Tiedemann delivered a new, more refined view of the absorption of nutrients through the gastrointestinal tract; they were the founders of modern physiology.
              • The mineralogical works of Gmelin were analyses of various minerals, such as the Hauyne with which he made his habilitation in Göttingen, or the Laumontite and the Cordierite. In addition, Gmelin also analysed mineral waters and in 1825 published the work try of a new chemical mineral system, since he knew that the time’s usual division on outer or physical characteristics was inadequate. Leopold Gmelin’s mineral system was taken largely critical among experts, but the basic idea of an order based on the chemical composition proved to be useful.
              • Gmelin released the Handbook of theoretical chemistry, which was continued as the Gmelin Handbook of Inorganic Chemistry until 1997 in about 800 volumes by the Gmelin Institute, and it is continued by the Gesellschaft Deutscher Chemiker as a database. The manual, even during his lifetime his most important work, was initially intended to be a textbook, which should unite the whole chemical knowledge at that time. Due to the enormous increase in knowledge and the associated development of the handbook into a reference book, Gmelin published a compact textbook of chemistry in 1844. His chemical achievements include the discovery of the Croconic acid; he thus had synthesised the first cyclic organic compound, and the previously mentioned discovery of the red prussiate.
              • Leopold Gmelin also developed a forerunner of the Periodic table and improved chemical equipment.
              • Chemische Untersuchung des schwarzen Pigments der Ochsen- und Kälberaugen, nebst einigen physiologischen Bemerkungen über dasselbe, Dissertation, Göttingen 1812, in Latein. Schweiggers Journ. 10, S. 507–547, 1814
              • Oryktognostische und chemische Beobachtungen über den Haüyn und einige mit ihm vorkommende Fossilien, nebst geognostischen Bemerkungen über die Berge des alten Latiums, Schweiggers Journ. 15 S. 1-41, 1815; Ann. Phil. Thomson 4, S. 115-122; 193-199, 1814
              • Leopold Gmelin, Friedrich Wöhler: Neue Cyanverbindungen, Schweiggers Journ. 36 S. 230–235, 1822
              • Versuch eines neuen chemischen Mineralsystems, Taschenbuch gesammte Mineralog. 19, I S. 322-334; 418-474; 490-507, 1825, II S. 33-77; 97-148, 1825
              • Friedrich Tiedemann, Leopold Gmelin: Die Verdauung nach Versuchen, Heidelberg und Leipzig 1826, 2 Bde.
              • Lehrbuch der Chemie zum Gebrauche bei Vorlesungen auf Universitäten, in Militärschulen, polytechnischen Anstalten, Realschulen etc. sowie zum Selbstunterrichte, Heidelberg, Universitätsbuchhandlung Karl Winter, 1844
            • The term biliverdin was coined by Jöns Jacob Berzelius in 1840, although he preferred “bilifulvin” (yellow/red) over “bilirubin” (red).
            • The term “bilirubin” was thought to have become mainstream based on the works of Staedeler in 1864 who crystallized bilirubin from cattle gallstones.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.][HIAN SIONG LEON MARIA TJEN (1944). “CHOLESCINTIGRAPHY the clinical application of 99m Technetium-diethyl-IDA to the investigation of the liver and biliary tract” (PDF). Archived (PDF) from the original on 3 November 2021.]}
            • Rudolf Virchow in 1847 recognized hematoidin to be identical to bilirubin.^{[Lightner DA (2013). “Early Scientific Investigations”. Bilirubin: Jekyll and Hyde Pigment of Life. Progress in the Chemistry of Organic Natural Products. Vol. 98. pp. 9–179. doi:10.1007/978-3-7091-1637-1_2. ISBN 978-3-7091-1636-4.]} It is not always distinguished from hematoidin, which one modern dictionary defines as synonymous with it^{[Merriam-Webster, Merriam-Webster’s Unabridged Dictionary, Merriam-Webster, archived from the original on 25 May 2020, retrieved 14 January 2018.]} but another defines as “apparently chemically identical with bilirubin but with a different site of origin, formed locally in the tissues from hemoglobin, particularly under conditions of reduced oxygen tension.”^{[Elsevier, Dorland’s Illustrated Medical Dictionary, Elsevier, archived from the original on 11 January 2014, retrieved 14 January 2018.][Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} 
            • The synonymous identity of bilirubin and hematoidin was confirmed in 1923 by Fischer and Steinmetz using analytical crystallography.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]}
            • In the 1930s, significant advances in bilirubin isolation and synthesis were described by Hans Fischer, Plieninger, and others,^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} and pioneering work pertaining to endogenous formation of bilirubin from heme was likewise conducted in the same decade.^{[Hopper, Christopher P.; Zambrana, Paige N.; Goebel, Ulrich; Wollborn, Jakob (2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. PMID 33838343. S2CID 233205099.]} The suffix IXα is partially based on a system developed Fischer, which means the bilin‘s parent compound was protoporphyrin IX cleaved at the alpha-methine bridge (see protoporphyrin IX nomenclature).^{[Berk, Paul D.; Berlin, Nathaniel I. (1977). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 27, 50.]}
            • Origins pertaining to the physiological activity of bilirubin were described by Ernst Stadelmann in 1891, who may have observed the biotransformation of infused hemoglobin into bilirubin possibly inspired by Ivan Tarkhanov‘s 1874 works.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} 
              • Ivan Romanovich Tarkhanov or Ivane Tarkhnishvili Georgian physiologist and science populariser from the Tarkhan-Mouravi noble family.^{[“Tarkhanov, Ivan Ramazovich”. TheFreeDictionary.com. Retrieved 21 January 2013.][Tsagareli, M. G. (November 13, 2010). “Ivane Tarkhnishvili: Major Georgian figure from Russian physiological school” (PDF). Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience. pp. 26–27. Archived from the original (PDF) on 2010-12-01. Retrieved 21 January 2013.][“Tarchanoff phenomenon”. TheFreeDictionary.com. Retrieved 21 January 2013.]} 
              • He led the Department of Physiology at the Academy of Military Medicine from 1877 to 1895 and authored a slew of articles on physiology for the Brockhaus and Efron Encyclopedic Dictionary.
              • Among his numerous contributions was the discovery of the skin galvanic reflex (1889). However, Tarkhnishvili’s most significant contribution was the discovery of the influence of X-rays on the central nervous system, animal behavior, the heart and circulation, and embryonic development (1896-1903). Indeed, these works have given rise to a new field in science as Radiobiology.
              •  His greatest interest was in electrophysiology, which was a direct continuation of the work of I. M. Sechenov, of whom Tarkhanov was one of the first disciples. Tarkhanov engaged in experimental studies on the phenomena of summation in the nervous system (1869). He also studied the influence of compressed air, oxygen, and carbonic acid on nervous irritability (1876). He described the formation of bile pigments in animals and humans (1874) and was one of the first to show (1871) the restoration of fading functions in anemic animals by infusing saline in the body. He dominated work in the field of the physiology of aging (1891) and many other topics. Tarkhanov was one of the first to investigate hypnotic suggestion. Tarkhanov’s books, Hypnotism, Suggestion and Mind-reading (1886; translated into French in 1891) and Suggestion and Hypnotism (1905) aroused wide public interest.
              • In 1885 experiments on cutting and artificial emptying of the seminal vesicles, Tarkhanov showed that the latter played the crucial role in the generation of sexual excitement in frogs. Proceeding from these experimental results, Tarkhanov put forward a hypothesis that filling and evacuation of the seminal vesicles were the main biological cause which led to sexual arousal and its disappearance in mammals and humans.^{[Тарханов И. Р. К физиологии полового аппарата у лягушки (On Physiology of the Reproductive system in frogs). — «Русская медицина (Russian medicine)», 1885, №30–32, с. 1–26. See also in German: Tarchanoff, J. R., Arch. f. d. g. Physiol. des Mensches u. d. Thierc., 40, 330 (1887).]}
              • Tarkhanov is probably best known as a pioneer of psychophysiology and radiobiology. In 1889, he was the first to observe and document the psychogalvanic reflex, i.e., variations in skin electrical potentials in the absence of any external stimuli. Tarkhanov’s method is still used today to measure skin potential. It records weak current actually produced by the body. Tarkhanov demonstrated that not only physical stimuli, but also mental activity, resulted in skin potential changes. The skin galvanic reflex is still used in applied psychophysiology as part of the polygraph in lie detection in which changes are recorded in several physiological variables while the subject is asked a series of questions pertaining to a specific issue under investigation.^{[Handbook of Clinical and Experimental Neuropsychology (eds. Gianfranco Denes, Luigi Pizzamiglio). Psychology Press, 1999. ISBN 9780863775420. Page 33.]}
              • After irradiating frogs and insects with X-rays in early 1896, several weeks after Röntgen‘s discovery, Tarkhanov concluded that these newly discovered rays not only photograph, but also “affect the living function”. These experiments signaled the birth of radiobiology.^{[ Y. B. Kudriashov. Radiation Biophysics. ISBN 9781600212802. Page xxi.]} 
              • Tarkhanov found a marked attenuation of excitability and a total suppression of acidic reflexes. These experiments confirmed that the impairment of reflexes after X-ray exposure depended on neither analgesia nor sensitive skin but on the moderating effect of the central nervous system (CNS) itself. Studying the effects of X-rays on metabolism in the myocardium and the circulation of the heart, he concluded that all of the effects of X-rays were due to their moderating or retarding the activity of the CNS (1896). A few years later, Tarkhanov presented an extensive paper on the role of X-rays in biology and medicine (1903). Thus, his pioneer works had indeed forecast a new field of science as radiobiology.
              • Tarkhanov worked intensively at translating many medical and physiology textbooks, among them Technical Textbook of Histology, by L.-A. Ranvier (1876) and General Muscle and Nerve Physiology by I. Rosenthal (1879). Between the years 1892 and 1904, Tarkhanov contributed nearly 160 articles, from B to Z, in physiology and medicine to the Brockhaus and Efron Encyclopedic Dictionary. Following his resignation from the St. Petersburg Military Medical Academy, he published during the period 1897–1908 nearly 250 popular articles on a variety of topics. In these publications, Tarkhanov discussed many exciting problems of the time, such as health, hygiene, and nutrition of the people, issues of education of children and women, the organization of women’s higher medical education in Russia, and radiation safety. He appears through his writings as a progressive humanist scholar, struggling for justice in all areas of public life and many others. His great capacity for work, which could not be reflected negatively in his health, is amazing.
              • Ivan R. Tarkhanov played an important role in Russian and European physiology. During his research and relatively short life, he established a school of physician-investigators of various specialties. From this school emerged eminent physiologists, including V. Y. Chagovets (1873–1941), B. F. Werigo (1860–1925), V. I. Vartanov (1853–1919), N. Cybulski (1854–1919), and V. K. Anrep (1852–1927).
              • Tarkhanov holds a special place in the history of Georgia, Georgian culture, and education. He was the first Georgian physiologist before Ivane Beritashvili, who was himself the second outstanding Georgian physiologist from the Russian physiological school. Tarkhanov (Tarkhnishvili) was one of those bridges, through which the people of Georgia joined with the best Russian and European science and culture, in searching for more advanced education, social progress, and independence.
              • Tarchanoff, J.R. Über die Bildung von Gallenpigment aus Blutfarbstoff im Thierkörper. Pflüg. Arch. ges. Phys., 1874, Bd. 9, 53-65.
              • Tarchanoff, J.R. Du rôle des vaisseaux capillaries dans la circulation. Compt. rend. Soc. de Biol. 1875, vol. 26, T. I, Sec. 6, 331-333.
              • Tarchanoff, J.R. Etude sur les centre psychomoteur des animaux nouveau – nès et sur leur dèveloppments dans différentes conditions. Compt. rend. Soc. de Biol. 1878, vol. 30, 217-221.
              • Tarchanoff, J.R. Über die willkürliche Acceleration der Herzschläge beim Menschen. Pflüg. Arch. ges. Phys., 1885, Bd. 35, 109-137.
              • Tarchanoff, J.R. Zur Phisiologie des Geschlechtsapparates des Frosches. Pflüg. Arch. ges. Physiol., 1887, .Bd. 40, 330-351.
              • Tarchanoff, J.R. Décharges èlectrique dans la peau de l’homme sous l’influence de l’excitation des organes des sens et de differentes formes d’activitè psychique. Compt. rend. Soc. de Biol. 1889, vol. 41, 447-451.
              • Tarchanoff, J.R. Hypnotisme, suggestion et lecture des pensèes. (Trad. par E. Jaubert). 1-er èd., Paris, 1891; II-me èd., Paris, 1893, Masson. 164 pp.
              • Tarchanoff, J.R. Quelques observations sur le sommeil normal. Arch. Italiennes de Biologie, 1894, vol. 21, 318-321.
              • Tarchanoff, J.R. Influence de la musique sur l’homme et sur les animaux. Arch. Italiennes de Biologie, 1894, vol. 21, 313-317.
              • Tarkhanov, I.R. Experiments upon the action of Roentgen’s X-rays on organisms. Izvestya. St.-Peterb. Biol. Lab., 1896, no.3, 47-52. (in Russian)
              • Tarchanoff, J.R. Actions physiologiques des tubes de Crookes à distance. Compt. rend. Soc. de Biol. 1897, vol. 49, 740-743.
              • Tarchanoff, I. “Dècapitation”. Dictionnaire de Physiologie, Ch. Rischet, 1898/1899, Paris, Baillièr, 681-691.
              • Tarkhanov, I.R. Soul and Body. 1904, St.-Petersburg, 176 pp. (in Russian)
              • Tarchanoff, J.R. et Moldenhauer, T. Sur la radio-activitè induite et naturelle des plantes et sur son rôle probable dans la croissance des plantes. Bull. Int. L’acad. Sci. Cracovie. 1905, no.1, 728-734.
              • Cybulski N. et Tarchanoff I. A propose des poisons normaux de l’intestine. Arch. Int. Physiol., 1907, vol. 5, 257-261.
            • Georg Barkan, Belarusian pharmacologist who taught at the Goethe University in Frankfurt. suggested the source of endogenous bilirubin to be from hemoglobin in 1932.^{[Barkan, Georg; Schales, Otto (1938). “A Hæmoglobin from Bile Pigment”. Nature. 142 (3601): 836–837. Bibcode:1938Natur.142..836B. doi:10.1038/142836b0. ISSN 1476-4687. S2CID 4073510.]} 
              • During the First World War , Barkan worked as a military doctor , later he was a department doctor in the flying school at the Lechfeld air base . From 1919 he worked as an assistant to Otto Frank in Munich and to Paul Morawitz in Würzburg . In 1923 he moved to Alexander Ellinger at Frankfurt University. From 1927 he was a private lecturer there. ^{[The fact that he has not yet held a professorship in Frankfurt is also documented by the files in the Hessian Main State Archives, in which he was only referred to as “Dr.” and “lecturer” appears. (see web links)]} In 1929 Barkan was granted leave of absence in Frankfurt ^{[German Society for Internal Medicine: Commemorating and remembering Georg Barkan . Heuer/Wolf do not mention this leave of absence, but Benzenhöfer also counts him among those persecuted by the Frankfurt University. (Udo Benzenhöfer: “The Frankfurt University Medicine between 1933 and 1945”, p. 37). The files in the Hessian Main State Archives in Wiesbaden, especially the reparations file that is available there, also speak in favor of the continued existence of Barkan’s ongoing employment relationship in Frankfurt.]} and moved to the University of Tartu in Estonia as full professor and director of the Pharmacological Institute .
              • In 1937 he was dismissed from the University of Tartu: The German Society for Internal Medicine states the following about the background to this : “The work as a German professor in Estonia was characterized by tensions against the background of Estonian nationalism and German-Baltic history. In 1937 Barkan was dismissed because he did not intend to learn the Estonian language.” ^{[ German Society for Internal Medicine: Commemorating and remembering Georg Barkan]}
              • Barkan returned to Germany via Switzerland . As a Jew, however, he no longer had any career opportunities there and emigrated in 1938 together with his wife Charlotte (nee Milch; 1894-1968) and his son Benedict Gunter (1925-2004) [ German Society for Internal Medicine: Commemorating and remembering Georg Barkan] to the USA, ^where he was a professor of biochemistry at Boston university .
              • His main research areas were blood pigments , iron metabolism and iodine pharmacology . He was co-editor of the Naunyn-Schmiedebergs Archiv .
              • On the question of stimulus conduction in the mammalian heart. royal Court book printers Kästner and Callwey, Munich 1914 (dissertation).
              • Iron Studies . De Gruyter , Berlin , Leipzig 1927 ( habilitation thesis ).
              • Procedure for determining the blood iron that can be easily switched off . Berlin, Vienna 1935.
              • Methods for studying the functions of the individual organs of the animal organism .
              • Renate Heuer , Siegbert Wolf (eds.): The Jews of Frankfurt University . Campus Verlag, Frankfurt am Main 1997, ISBN 3-593-35502-7 , p. 24 f .
              • Udo Benzenhöfer : “The Frankfurt University Medicine between 1933 and 1945”, Klemm + Oelschläger, Münster 2012, ISBN 978-3-86281-050-5 ( full text ).
              • German Society for Internal Medicine: Commemorating and Remembering Georg Barkan .
              • Hessian Main State Archive Wiesbaden: Compensation procedure Georg Barkan, signature: HHStAW inventory 518 no. 46754; Files of the Hessian Ministry of Culture: case file Georg Barkan: signature: HHStAW inventory 504 no. 12040.
              • Barcan, George. Hessian biography. (As of March 3, 2023). In: State Historical Information System Hesse (LAGIS).
            • Plieninger and Fischer demonstrated an enzymatic oxidative loss of the alpha-methine bridge of heme resulting in a bis-lactam structure in 1942.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} 
              • Plieninger
                • German chemist
                • doctorate in 1941 (on the constitution of bilirubin and on tripyrrene ) with Hans Fischer . ^{[ The tripyrrenes were a subject of Fischer’s research. See : Hans Fischer; Herbert Reinecke: About tripyrrenes , in: Hoppe-Seyler’s journal for physiological chemistry , volume 259, 1939, p. 83 ff.]} 
                • He did military service from 1935 to 1937 as well as military service in 1939/40 and 1945. During the rest of the Second World War he was partly a research assistant at the Munich Technical University (where Hans Fischer managed to find him indispensable in 1940 and 1945 )
                • and from 1942 to 1944 he worked at the IG Farben Ludwigshafen at Walter Repp . 
                • From 1946 to 1953 he worked for Knoll AG in Ludwigshafen, where he worked on the synthesis of amino acids.
                •  In 1953 he habilitated with his already published work at the TH Darmstadt. For formal reasons, he was not able to habilitate in Heidelberg, since Karl Freudenberg , his father-in-law, was the director of the institute there.
                • He was at the Ruprecht-Karls-University Heidelberg from 1953 assistant, from 1958 nutritionist, from 1960 ^adjunct professor , from 1964 associate and from 1967 full professor . He was twice executive director of the chemical institute.
                • He dealt with organic synthesis especially in natural products.
                • In 1956 he published the dienol-benzene rearrangement with Rolf Müller . In 1962 he introduced high-pressure processes to preparative organic synthesis.
                • He was married to Karl Freudenberg ‘s daughter Herta . He also edited new editions of Freudenberg’s Textbook of Organic Chemistry .
                • He and his wife were involved in environmental protection and peace politics. In 1982, for example, he sent all 519 members of the Bundestag the book The Fate of the Earth by Jonathan Schell against nuclear armament at his own expense (around 10,000 DM at the time) . He had a farm in Tuscany where he practiced organic farming.
              • Fischer
                • German organic chemist and the recipient of the 1930 Nobel Prize for Chemistry “for his researches into the constitution of haemin and chlorophyll and especially for his synthesis of haemin.”^{[ “The Nobel Prize in Chemistry 1930”. nobelprize.org.]}
                • He worked first at a Medical Clinic in Munich and then at the First Berlin Chemical Institute under Emil Fischer (who shows up here regularly). He returned to Munich in 1911 and qualified as lecturer on internal medicine one year later. In 1913, he became a lecturer in physiology at the Physiological Institute in Munich. In 1916, he became Professor of Medical Chemistry at the University of Innsbruck and from there he went to the University of Vienna in 1918.
                • From 1921 until his death, he held the position of Professor of Organic Chemistry at the Technical University of Munich.
                • Fischer’s scientific work was mostly concerned with the investigation of the pigments in blood, bile, and also chlorophyll in leaves, as well as with the chemistry of pyrrole from which these pigments are derived. Of special importance was his synthesis of bilirubin and haemin.
                • He received many honors for this work, and received the Nobel Prize in 1930.
                  • Fellow of the Academy of Sciences Leopoldina (1919)
                  • Privy Councillor (1925)
                  • Liebig Memorial Medal (1929)
                  • Nobel Prize for Chemistry (1930)
                  • Honorary doctorate, Harvard University (1936)
                  • Davy Medal of the Royal Society of London (1937)
                • The lunar crater Fischer was named after him (and Hermann Emil Fischer) in 1976.
                • Hans Fisher had mapped the composition of a hem group. In 1929 Fischer succeeded in producing the substance and proving that its ring has a central atom of iron, as he also continued studying other pigmented substances of a biological importance of biochemistry such as chlorophyll, the color that plays part in a plants photosynthesis.
                • Fischer also unraveled the bile pigments biliverdin (which causes the yellowish color characteristic of bruised skin) and bilirubin (which yellows skin in jaundice cases), and synthesized them in 1942 and 1944, successively.
                • He conducted microanalyses of more than 60,000 chemical substance, and had won the Nobel Prize for Chemistry in 1930.
                • The person that sparked Fischer’s interest was von Muller, his former professor and supervisor whom interests were in pyrrole pigments by inviting Fischer to work with him at the well-known Second Medical Clinic in Munich in 1910. Under Muller, he began to examine the composition of the bile pigment bilirubin, something he would continue to be engaged in during the decades that followed. Fischer’s succession also came with difficulty as many of his experiments seemed to have failed but with time Fischer was able to perfect his acknowledgement with is failed attempts.
                • Fischer married Wiltrud Haufe around his 50’s in the year 1935. Fischer was a man whom dedicated almost “exclusively” to his work. He continued his scientific research during Germany’s Nazi era, and committed suicide on Easter Sunday in 1945, after his laboratory and life’s work had been destroyed by the bombing in the last days of World War II.
                • Heinrich Wieland (1950), “Hans Fischer und Otto Hönigschmid zum Gedächtniss”, Angewandte Chemie, 62 (1): 1–4, doi:10.1002/ange.19500620102.
                • Bickel, M H (2001), “[Henry E. Sigerist and Hans Fischer as pioneers of a medical history institute in Zurich]”, Gesnerus, vol. 58, no. 3–4, pp. 215–9, PMID 11810971
                • Stern, A J (1973), “Hans Fischer (1881–1945)”, Ann. N. Y. Acad. Sci., vol. 206, no. 1, pp. 752–61, Bibcode:1973NYASA.206..752S, doi:10.1111/j.1749-6632.1973.tb43252.x, PMID 4584221, S2CID 40633114
                • Watson, C J (1965), “Reminiscences of Hans Fischer and his laboratory”, Perspect. Biol. Med., vol. 8, no. 4, pp. 419–35, doi:10.1353/pbm.1965.0052, PMID 5323649, S2CID 32016198
                • Kämmerer, H (1961), “Hans Fischer (1881–1945). A reminiscence on the 80th anniversary of his birth”, Münchener Medizinische Wochenschrift (1950) (published Nov 3, 1961), vol. 103, pp. 2164–6, PMID 14036988
                • Newspaper clippings about Hans Fischer in the 20th Century Press Archives of the ZBW 
                • Hans Fischer on Nobelprize.org  including the Nobel Lecture, December 11, 1930
            • It is widely accepted that Irving London was the first to demonstrate endogenous formation of bilirubin from hemoglobin in 1950,^{[ “Bilirubin”. American Chemical Society. Retrieved 28 May 2021.]} 
              • Irving M. London (July 24, 1918 – May 23, 2018) was a hematologist and geneticist. He was an associate professor of medicine at Columbia University College of Physicians and Surgeons when he was selected to be the founding chair of the department of medicine at the Albert Einstein College of Medicine in 1955.^{[“Einstein Medical Heads – Three Department Chairmen Named, Filling Senior Faculty”. New York Times. March 21, 1955. p. 26. Retrieved 17 January 2016.]} He was recruited to become the founding director of the Harvard-MIT Program in Health Sciences and Technology in 1970.^{[“Web Pages of the Harvard-MIT HST Program”. Archived from the original on 2016-03-05. Retrieved 2015-12-25.][ “Dr. Irving London to be Honored at Harvard”. Albert Einstein College of Medicined. Retrieved November 11, 2021.]} Dr. London was the first professor to hold dual roles at both Harvard and MIT.^{[“School Marks Pioneer’s Passing”. hms.harvard.edu. Retrieved 2021-05-28.]}
              • London graduated from Harvard College and Harvard Medical School.^{[ “Irving M. London”.]} 
              • London graduated from Harvard College in 1939 summa cum laude. He was on a student committee at Harvard that gave 14 refugee students the opportunity to leave Nazi-occupied Europe to study in Boston.^{[“School Marks Pioneer’s Passing”. hms.harvard.edu. Retrieved 2021-05-28.]} London also earned a second undergraduate degree from Hebrew College in Roxbury at the same time. London delivered the graduating address at Harvard, the content of which was inspired by his thesis “The Jeffersonian Tradition in American Nationalism”. London gave serious though to attending law school after graduation, but ultimately chose to enroll in medical school.^{[“Irving M. London”. meded.hms.harvard.edu. Retrieved 2021-05-29.]}
              • After completing an MD from HMS in 1943, Dr. London accepted an intership at Columbia-Presbyterian Medical Center in New York. During World War II he served as a US Army captain in the Medical Corps were he conducted research on the use of chloroquine as an antimalarial medication. After the war, he was assigned to Bikini Atoll in the Marshall Islands of the South Pacific Ocean to serve a physician at the atomic bomb testing.^{[ “Remembering Dr. Irving M. London, Founding Director of the Harvard-MIT Health Sciences and Technology program”. Institute for Medical Engineering & Science. 2018-05-24. Retrieved 2021-05-28.]}
              • London returned to New York City after the war to continue residency training. Upon completion, he joined the department of biochemistry at Columbia University College of Physicians & Surgeons and was promoted to faculty, teaching and tenure. His research focused on the lifespan of red blood cells in normal and pathological conditions.^{[ “School Marks Pioneer’s Passing”. hms.harvard.edu. Retrieved 2021-05-28.]} In 1954, he was selected to be the founding chair of the department of medicine at the Albert Einstein College of Medicine, and was director of medical services at Bronx Municipal Hospital until 1970.^{[“Remembering Dr. Irving M. London, Founding Director of the Harvard-MIT Health Sciences and Technology program”. Institute for Medical Engineering & Science. 2018-05-24. Retrieved 2021-05-28.]}
              • In 1968, London was invited as a consultant to planning for the Massachusetts Institute of Technology and Harvard Medical School joint program. In 1970 he accepted a position a director of the new Harvard-MIT Program in Health Sciences and Technology, and around 1972 he was also a physician at Peter Bent Brigham Hospital.^{[ “Med School, MIT Assign Dual Post To Irving London | News | The Harvard Crimson”. www.thecrimson.com. Retrieved 2021-05-29.]} London served as director of the program until 1985 while simultaneously a professor of medicine at HMS and a professor of biology at MIT.^{[ “Remembering Dr. Irving M. London, Founding Director of the Harvard-MIT Health Sciences and Technology program”. Institute for Medical Engineering & Science. 2018-05-24. Retrieved 2021-05-28.]}
              • London is best known for groundbreaking explanation for the molecular regulation (gene transcription and translation) of hemoglobin synthesis.^{[ “Remembering Dr. Irving M. London, Founding Director of the Harvard-MIT Health Sciences and Technology program”. Institute for Medical Engineering & Science. 2018-05-24. Retrieved 2021-05-28.]} London and colleagues demonstrated that hemoglobin is the endogenous source of bilirubin,^{[“Bilirubin”. American Chemical Society. Retrieved 2021-05-28.]} an important event in the fields of jaundice and heme oxygenase research.
              • London died on May 23, 2018, two months before his 100th birthday.^{[“Irving London, founding director of Harvard-MIT Program in Health Sciences and Technology, dies at 99”. MIT News. 25 May 2018.]}
              • Welch Fellowship in Internal Medicine of the National Academy of Sciences 1949-1952
              • Theobald Smith Award in Medical Sciences of the American Association for the Advancement of Science 1953
              • Commonwealth Fund Fellowship at Institut Pasteur 1962-1963
              • election to American Academy of Arts and Sciences 1963
              • charter member in the Institute of Medicine of the National Academy of Sciences in 1970
              • elected member National Academy of Science 1971
              • board of directors for Biosciences Advisory Committee for Johnson & Johnson 1982-2003
              • establishment of The Irving M. London Society (HST) at Harvard Medical School^[7]
              • The Dr. Irving M. London Teaching Award, initiated in 1986^{[“Community Awards”. Harvard-MIT Health Sciences and Technology. 2019-12-19. Retrieved 2021-05-29.]}
            • Sjostrand demonstrated hemoglobin catabolism produces carbon monoxide between 1949 and 1952.
              • Sjöstrand later demonstrated CO production from hemoglobin decomposition in 1952.^{[Hopper CP, Zambrana PN, Goebel U, Wollborn J (June 2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. ISSN 1089-8603. PMID 33838343. S2CID 233205099.]}
            • 14C labeled protoporphyrin biotransformation to bilirubin evidence emerged in 1966 by Cecil Watson.^{[Watson, Cecil J. (1977). “Historical Review of Bilirubin Chemistry”. In Berk, Paul D. (ed.). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 3–16.]} 
              • Cecil James Watson (May 31, 1901 – April 11, 1983) was an American hepatologist.^{[ Schmid, R (1994). Biographical Memoirs: Volume 65, Chapter 19, Cecil James Watson. NAP.edu: NAP.edu. pp. 354–406. doi:10.17226/4548. ISBN 978-0-309-05037-1.]}
              • Watson started his medical career at a clinic in Minot, North Dakota before moving to Germany in 1930 to work with Hans Fischer.^{[ Watson, C. J. (1965). “Reminiscences of Hans Fischer and His Laboratory”. Perspectives in Biology and Medicine. 8 (4): viii, 418–435. doi:10.1353/pbm.1965.0052. ISSN 1529-8795. PMID 5323649.]} 
              • Upon his return to the United States in 1932, Watson began working at Minneapolis General Hospital. By 1934, Watson was assistant professor of medicine at UM. Watson lead the medical school as chairman from 1943 to 1966, stepping down for a position at Northwestern Hospital.^{[Schmid, Rudi. “Cecil James Watson 1901—1983” (PDF). Biographical Memoirs of the National Academy of Sciences.]}
              • Watson was named a member of the National Academy of Sciences in 1959,^{[“Cecil J. Watson”. National Academy of Sciences. Retrieved October 15, 2018.]} and the Cecil J. Watson Award was inaugurated in his honor by the Minneapolis Society of Internal Medicine in 1961.^{[“Cecil J. Watson Award”. University of Minnesota. Retrieved October 15, 2018.]}
              • Watson died on April 11, 1983, aged 82.^{[ “Dr. Cecil J. Watson, 82, Dies; Expert on Disorders of Liver”. New York Times. United Press International. April 14, 1983. Retrieved October 15, 2018.]}
            • Rudi Schmid and Tenhunen discovered heme oxygenase, the enzyme responsible, in 1968.^{[Hopper, Christopher P.; Zambrana, Paige N.; Goebel, Ulrich; Wollborn, Jakob (2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. PMID 33838343. S2CID 233205099.]} 
              • Swiss-born American medical researcher specializing in hepatology. Among his contributions to biomedical science, Schmid led a team to discover heme oxygenase.^{[Tenhunen, R.; Marver, H. S.; Schmid, R. (1968-10-01). “The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase”. Proceedings of the National Academy of Sciences. 61 (2): 748–755. Bibcode:1968PNAS…61..748T. doi:10.1073/pnas.61.2.748. ISSN 0027-8424. PMC 225223. PMID 4386763.]}
                • HMOX1 was first characterized by Tenhunen and Rudi Schmid upon demonstrating it as the enzyme responsible for catalyzing biotransformation of heme to bilirubin.^{[Hopper CP, Zambrana PN, Goebel U, Wollborn J (June 2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. ISSN 1089-8603. PMID 33838343. S2CID 233205099.]}
                • Several labs attempted to explain the biotransformation of heme to biliverdin such as Nakajima et al. in 1962 who characterized a soluble “heme α-methenyl oxygenase”, however the findings could not be reproduced and alternative non-enzymatic explanations for their observation emerged. The earliest evidence of oxidative enzymatic biotransformation of heme to a bilin was demonstrated by Hans Plieninger and Hans Fischer in 1942.^{[Watson C (1977). “Historical Review of Bilirubin Chemistry”. In Berk P (ed.). Chemistry and Physiology of Bile Pigments. p. 5.]} The discovery of HMOX is a case of academic linage as Fischer was the academic adviser for Cecil Watson, and Watson was an adviser for Rudi Schmid.
                • Felix Hoppe-Seyler coined the name “haemoglobin”; haem being derived from Greek meaning blood, and globin from Latin globus meaning round object (see also: carboxyhemoglobin etymology). Hemoglobin was first discovered in the 1840s by Friedrich Ludwig Hünefeld.^{[Clegg B (2011). “Haemoglobin”. Chemistry World. Retrieved 2021-05-26. ^][Boor AK (January 1930). “A Crystallographic Study of Pure Carbonmon-Oxide Hemoglobin”. The Journal of General Physiology. 13 (3): 307–316. doi:10.1085/jgp.13.3.307. PMC 2141039. PMID 19872525.]} Heme (as hemin coordinated with chlorine) was characterized by Ludwik Karol Teichmann in 1853. Many labs investigated in vitro transformation of heme into bilins throughout the 1930s exemplified by the work of Georg Barkan,^{[Barkan G, Schales O (November 1938). “A Hæmoglobin from Bile Pigment”. Nature. 142 (3601): 836–837. Bibcode:1938Natur.142..836B. doi:10.1038/142836b0. ISSN 1476-4687. S2CID 4073510.]} followed by Esther Killick who recognized a presence of carbon monoxide to correlate with pseudohemoglobin (an obsolete bilin term coined by Barkan) in 1940.^{[Hopper CP, Zambrana PN, Goebel U, Wollborn J (June 2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. ISSN 1089-8603. PMID 33838343. S2CID 233205099.]} The endogenous biotransformation of heme to bilirubin is thought to have been definitively demonstrated with experimental evidence by Irving London in 1950,^{[ “Bilirubin”. American Chemical Society. Retrieved 2021-10-19.]} although trace evidence for the endogenous formation of bilirubin has origins dating back several centuries in the context of jaundice with innumerable global contributions (see also: History of Bilirubin).^{[Wegiel B, Otterbein LE (2012). “Go green: the anti-inflammatory effects of biliverdin reductase”. Frontiers in Pharmacology. 3: 47.][Watson C (1977). “Historical Review of Bilirubin Chemistry”. In Berk P (ed.). Chemistry and Physiology of Bile Pigments. p. 5.]}
                • CO was detected in exhaled breath 1869. Felix Hoppe-Seyler developed the first qualitative carboxyhemoglobin test, and Josef von Fodor developed the first quantitative analytical test for carboxyhemoglobin. The first reported detection of naturally occurring CO in human blood occurred in 1923 by Royd Ray Sayers et al. although they discarded their data as random error.^{[Hopper CP, Zambrana PN, Goebel U, Wollborn J (June 2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. ISSN 1089-8603. PMID 33838343. S2CID 233205099.]} Alexander Gettler confirmed CO to have a normal presence in blood in 1933, however, he attributed the finding to inevitable pollution exposure or perhaps derived from the human microbiome.^{[Hopper CP, De La Cruz LK, Lyles KV, Wareham LK, Gilbert JA, Eichenbaum Z, et al. (December 2020). “Role of Carbon Monoxide in Host-Gut Microbiome Communication”. Chemical Reviews. 120 (24): 13273–13311. doi:10.1021/acs.chemrev.0c00586. PMID 33089988. S2CID 224824871.]} Sjöstrand later demonstrated CO production from hemoglobin decomposition in 1952.^{[Hopper CP, Zambrana PN, Goebel U, Wollborn J (June 2021). “A brief history of carbon monoxide and its therapeutic origins”. Nitric Oxide. 111–112: 45–63. doi:10.1016/j.niox.2021.04.001. ISSN 1089-8603. PMID 33838343. S2CID 233205099.]}
              • Schmid was a postdoctoral researcher at Columbia University, then worked for the National Institutes of Health before joining the Thorndike Memorial Laboratory, a division of the Harvard Medical Unit at Boston City Hospital. He began teaching at the University of Chicago in 1962. Four years later, Schmid joined the UCSF faculty. He was dean of the UCSF School of Medicine from 1983 to 1989.^{[Ockner, Robert K. “Rudi Schmid 1922–2007” (PDF). Biographical Memoirs of the National Academy of Sciences.]}
              • Over the course of his career, Schmid was granted membership into the National Academy of Sciences, the Institute of Medicine, and the Academy of Sciences Leopoldina.^{[Chang, You-De (28 December 2007). “The love of a beloved hapatologist, Dr. Rudi Schmid”. World Journal of Gastroenterology. 13 (48): 6612–6613. doi:10.3748/wjg.v13.i48.6612. PMC 4611307.]} He was later honored with the establishment of the Rudi Schmid Distinguished Professorship in Neurology at UCSF.^{[ “Endowed Chairs and Distinguished Professorships”. University of California, San Francisco. Retrieved 14 October 2018.]}
              • Schmid died of pulmonary failure on 20 October 2007, at home in Kentfield, California, aged 85.^{[Allday, Erin (3 November 2007). “Rudi Schmid, former dean, UCSF School of Medicine”. San Francisco Chronicle. Retrieved 14 October 2018.]}
            • Earlier in 1963, Nakajima described a soluble “heme alpha-methnyl oxygeanse” which what later determined to be a non-enzymatic pathway, such as formation of a 1,2-Dioxetane intermediate at the methine bridge resulting in carbon monoxide release and biliverdin formation.^{[Berk, Paul D.; Berlin, Nathaniel I. (1977). International Symposium on Chemistry and Physiology of Bile Pigments. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health. pp. 27, 50.]}
            • Notable people
              • Claudio Tiribelli, Italian hepatologist, studies on bilirubin
            • See also:
              • Babesiosis, Biliary atresia, Bilirubin diglucuronide, Biliverdin, Crigler–Najjar syndrome, Gilbert’s syndrome, Lumirubin, Primary biliary cirrhosis, Primary sclerosing cholangitis
            • Hy’s Law
              • Hy’s law is a rule of thumb that a patient is at high risk of a fatal drug-induced liver injury if given a medication that causes hepatocellular injury (not Hepatobiliary injury) with jaundice.^{[Reuben, Adrian (March 26, 2008). “Hy’s Law Explained” (PDF). fda.gov. Retrieved December 7, 2016.]} The law is based on observations by Hy Zimmerman, a major scholar of drug-induced liver injury.^{[ United States Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER): Guidance for Industry Drug-Induced Liver Injury: Premarketing Clinical Evaluation, Final, July 2009][Robles-Diaz M, Lucena MI, Kaplowitz N, Stephens C, Medina-Cáliz I, González-Jimenez A, Ulzurrun E, Gonzalez AF, Fernandez MC, Romero-Gómez M, Jimenez-Perez M, Bruguera M, Prieto M, Bessone F, Hernandez N, Arrese M, Andrade RJ (July 2014), “Use of Hy’s law and a new composite algorithm to predict acute liver failure in patients with drug-induced liver injury”, Gastroenterology, 147 (1): 109–118, doi:10.1053/j.gastro.2014.03.050, PMID 24704526][ Tansel A, Kanwal F, Hollinger FB (Aug 2015), “Use of Hy’s Law, R criteria, and nR criteria to predict acute liver failure or transplantation in patients with drug-induced liver injury.”, Gastroenterology, 148 (2): 452, doi:10.1053/j.gastro.2014.11.046, PMID 25532807]} Some have suggested the principle be called a hypothesis or observation.^{[Senior, John R. (22 March 2006), “How can ‘Hy’s law’ help the clinician?”, Pharmacoepidemiology and Drug Safety, 15 (4): 235–239, doi:10.1002/pds.1210, PMID 16552792]}
              • Hy’s Law cases have three components:^{[United States Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER): Guidance for Industry Drug-Induced Liver Injury: Premarketing Clinical Evaluation, Final, July 2009]}
                • The drug causes hepatocellular injury, generally defined as an elevated ALT or AST by 3-fold or greater above the upper limit of normal. Often with aminotransferases much greater (5-10x) the upper limit of normal.
                • Among subjects showing such aminotransferase elevations, they also have elevation of their serum total bilirubin of greater than 2× the upper limit of normal, without findings of cholestasis (defined as serum alkaline phosphatase activity less than 2× the upper limit of normal).
                • No other reason can be found to explain the combination of increased aminotransferase and serum total bilirubin, such as viral hepatitis, alcohol abuse, ischemia, preexisting liver disease, or another drug capable of causing the observed injury.^{[Reuben, Adrian (March 26, 2008). “Hy’s Law Explained” (PDF). fda.gov. Retrieved December 7, 2016.]}
              • In Zimmerman’s analysis of 116 patients with hepatocellular injury and jaundice due to drug exposure, 76% went on to either require a liver transplant or died.^{[ Zimmerman, Hyman (1999). Hepatotoxicity. ISBN 978-0781719520.]} Other studies have reported a lower but still significant mortality of 10%.^{[Andrade, Raúl J.; Lucena, M. Isabel; Fernández, M. Carmen; Pelaez, Gloria; Pachkoria, Ketevan; García-Ruiz, Elena; García-Muñoz, Beatriz; González-Grande, Rocio; Pizarro, Angeles (2005-08-01). “Drug-induced liver injury: an analysis of 461 incidences submitted to the Spanish registry over a 10-year period”. Gastroenterology. 129 (2): 512–521. doi:10.1016/j.gastro.2005.05.006. ISSN 0016-5085. PMID 16083708.][Björnsson, Einar; Olsson, Rolf (2005-08-01). “Outcome and prognostic markers in severe drug-induced liver disease”. Hepatology. 42 (2): 481–489. doi:10.1002/hep.20800. ISSN 0270-9139. PMID 16025496.]}
  • blood:
    • Bilirubin+Albumin
  • liver:
    • Bilirubin glucuronide
      • Bilirubin glucuronide is a water-soluble reaction intermediate over the process of conjugation of indirect bilirubin.^{[Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.]} Bilirubin glucuronide itself belongs to the category of conjugated bilirubin along with bilirubin di-glucuronide.^{[Dubin-Johnson syndrome is associated with inability of the hepatocytes to secrete conjugated bilirubin after it has been formed.]} However, only the latter one is primarily excreted into the bile in the normal setting.^{[ Dubin-Johnson syndrome is associated with inability of the hepatocytes to secrete conjugated bilirubin after it has been formed.][Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.][Nishida, T; Gatmaitan, Z; Roy-Chowdhry, J; Arias, I M (1992-11-01). “Two distinct mechanisms for bilirubin glucuronide transport by rat bile canalicular membrane vesicles. Demonstration of defective ATP-dependent transport in rats (TR-) with inherited conjugated hyperbilirubinemia”. Journal of Clinical Investigation. American Society for Clinical Investigation. 90 (5): 2130–2135. doi:10.1172/jci116098. ISSN 0021-9738. PMC 443282. PMID 1430236.][Zhou, J.; Tracy, T. S.; Remmel, R. P. (2010-07-28). “Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1”. Drug Metabolism and Disposition. 38 (11): 1907–1911. doi:10.1124/dmd.110.033829. ISSN 0090-9556. PMC 2967393. PMID 20668247.[Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.]}
      • Upon macrophages spot and phagocytize the effete Red Blood Corpuscles containing hemoglobin,^{[Berk, Paul D.; Howe, Robert B.; Bloomer, Joseph R.; Berlin, Nathaniel I. (1969-11-01). “Studies of bilirubin kinetics in normal adults”. The Journal of Clinical Investigation. 48 (11): 2176–2190. doi:10.1172/jci106184. ISSN 0021-9738. PMC 297471. PMID 5824077.]} unconjugated bilirubin is discharged from macrophages into the blood plasma.^{[“Heme metabolism in macrophages”. eClinpath. Archived from the original on 2018-05-17. Retrieved 2019-05-05.][“Bilirubin and hemolytic anemia”. eClinpath. Archived from the original on 2018-08-07. Retrieved 2019-05-05.]} Most often, the free and water-insoluble unconjugated bilirubin which has an internal hydrodren bonding will bind to albumin and, to a much lesser extent, high density lipoprotein in order to decrease its hydrophobicity and to limit the probability of unnecessary contact with other tissues^{[Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.]} and keep bilirubin in the vascular space from traversing to extravascular space including brain, and from ending up increasing glomerular filtration. Nevertheless, there is still a little portion of indirect bilirubins stays free-of-bound. Free unconjugated bilirubin can poison the cerebrum.^{[Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.]}
        • Shapiro, Steven M. (January 2005). “Definition of the Clinical Spectrum of Kernicterus and Bilirubin-Induced Neurologic Dysfunction (BIND)”. Journal of Perinatology. 25 (1): 54–59. doi:10.1038/sj.jp.7211157. PMID 15578034. S2CID 19663259.
        • Brites, Dora (2012-05-29). “The Evolving Landscape of Neurotoxicity by Unconjugated Bilirubin: Role of Glial Cells and Inflammation”. Frontiers in Pharmacology. 3: 88. doi:10.3389/fphar.2012.00088. ISSN 1663-9812. PMC 3361682. PMID 22661946.
        • Wusthoff, Courtney J.; Loe, Irene M. (2015-01-10). “Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes”. Seminars in Fetal & Neonatal Medicine. 20 (1): 52–57. doi:10.1016/j.siny.2014.12.003. ISSN 1744-165X. PMC 4651619. PMID 25585889.
        • Radmacher, Paula G; Groves, Frank D; Owa, Joshua A; Ofovwe, Gabriel E; Amuabunos, Emmanuel A; Olusanya, Bolajoko O; Slusher, Tina M (2015-04-01). “A modified Bilirubin-induced neurologic dysfunction (BIND-M) algorithm is useful in evaluating severity of jaundice in a resource-limited setting”. BMC Pediatrics. 15 (1): 28. doi:10.1186/s12887-015-0355-2. ISSN 1471-2431. PMC 4389967. PMID 25884571.
        • Johnson, Lois; Bhutani, Vinod K. (2011). “The Clinical Syndrome of Bilirubin-Induced Neurologic Dysfunction”. Seminars in Perinatology. 35 (3): 101–113. doi:10.1053/j.semperi.2011.02.003. ISSN 0146-0005. PMID 21641482.
        • Bhutani, Vinod K.; Wong, Ronald (2015). “Bilirubin-induced neurologic dysfunction (BIND)”. Seminars in Fetal and Neonatal Medicine. 20 (1): 1. doi:10.1016/j.siny.2014.12.010. ISSN 1744-165X. PMID 25577656.
        • Press, Dove (2018-03-07). “Acute bilirubin encephalopathy and its progression to kernicterus: cur – RRN”. Research and Reports in Neonatology. 8: 33–44. doi:10.2147/RRN.S125758. Archived from the original on 2019-05-06. Retrieved 2019-05-06.
        • Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4. However, when jaundice is present it is likely that many other potentially toxic materials have also accumulated in the blood as a consequence of their reflux from the bile or impaired secretion from the hepatocyte. This can lead to impaired mental function and malaise.
        • Watchko, Jon F.; Tiribelli, Claudio (2013-11-21). Ingelfinger, Julie R. (ed.). “Bilirubin-Induced Neurologic Damage — Mechanisms and Management Approaches”. The New England Journal of Medicine. 369 (21): 2021–2030. doi:10.1056/nejmra1308124. ISSN 0028-4793. PMID 24256380.
        • Shapiro, Steven M.; Bhutani, Vinod K.; Johnson, Lois (2006). “Hyperbilirubinemia and Kernicterus”. Clinics in Perinatology. 33 (2): 387–410. doi:10.1016/j.clp.2006.03.010. ISSN 0095-5108. PMID 16765731
      • Finally, albumin leads the indirect bilirubin to the liver.^{[Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.][Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.]} In the liver sinusoid, albumin disassociates with the indirect bilirubin and returns to the circulation while the hepatocyte transfers the indirect bilirubin to ligandin and glucuronide conjugates the indirect bilirubin in the endoplasmic reticulum by disrupting unconjugated bilirubin’s internal hydrogen bonding, which is the thing that makes indirect bilirubin having the property of eternal half-elimination life and insoluble in water,^{[Structure of bilirubin Bonnet RJ, Davis E, Hursthouse MB Nature. 1976; 262:326.][Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.][Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.][The Liver: Biology and Pathobiology. Hoboken, N.J: Wiley. 2013. ISBN 978-1-119-96422-3. OCLC 899743347.][“Hereditary Jaundice and Disorders of Bilirubin Metabolism – The Online Metabolic and Molecular Bases of Inherited Disease – McGraw-Hill Medical”. OMMBID. 2019-05-06. Retrieved 2019-05-06.]} and by attaching two molecules of glucuronic acid to it in a two step process.^{[“Bilirubin”. Spencer S. Eccles Health Sciences Library Home Page. 2019-05-06. Archived from the original on 2019-05-06. Retrieved 2019-05-06.]} The reaction is a transfer of two glucuronic acid groups including UDP glucuronic acid sequentially to the propionic acid groups of the bilirubin, primarily catalyzed by UGT1A1.^{[“Bilirubin”. Spencer S. Eccles Health Sciences Library Home Page. 2019-05-06. Archived from the original on 2019-05-06. Retrieved 2019-05-06.][ Bilirubin must be conjugated to a water-soluble substance][Zhou, J.; Tracy, T. S.; Remmel, R. P. (2010-07-28). “Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1”. Drug Metabolism and Disposition. 38 (11): 1907–1911. doi:10.1124/dmd.110.033829. ISSN 0090-9556. PMC 2967393. PMID 20668247.]} In greater detail about this reaction, a glucuronosyl moiety is conjugated to one of the propionic acid side chains, located on the C8 and C12 carbons of the two central pyrrole rings of bilirubin.^{[Kadakol, A; Ghosh, SS; Sappal, BS; Sharma, G; Chowdhury, JR; Chowdhury, NR (2000). “Genetic lesions of bilirubin uridine-diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler-Najjar and Gilbert syndromes: correlation of genotype to phenotype”. Human Mutation. 16 (4): 297–306. doi:10.1002/1098-1004(200010)16:4<297::AID-HUMU2>3.0.CO;2-Z. ISSN 1059-7794. PMID 11013440. S2CID 24275067.]} When the first step is completely done, the substrate bilirubin glucuronide (also known as mono-glucuronide^{[ Crawford, JM; Ransil, BJ; Narciso, JP; Gollan, JL (1992-08-25). “Hepatic microsomal bilirubin UDP-glucuronosyltransferase. The kinetics of bilirubin mono- and diglucuronide synthesis”. The Journal of Biological Chemistry. 267 (24): 16943–50. doi:10.1016/S0021-9258(18)41876-5. ISSN 0021-9258. PMID 1512236.]}) is born at this stage and is water-soluble and readily excreted in bile.^{[Bilirubin must be conjugated to a water-soluble substance][Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.]} Thereafter, so long as the second step of attachment of the other glucuronic acid to it succeeds (officially called “re-glucuronidated“), the substrate bilirubin glucuronide will turn into bilirubin di-glucuronide (8,12-diglucuronide^{[Crawford, JM; Ransil, BJ; Narciso, JP; Gollan, JL (1992-08-25). “Hepatic microsomal bilirubin UDP-glucuronosyltransferase. The kinetics of bilirubin mono- and diglucuronide synthesis”. The Journal of Biological Chemistry. 267 (24): 16943–50. doi:10.1016/S0021-9258(18)41876-5. ISSN 0021-9258. PMID 1512236.]}) and be excreted into bile canaliculi by way of C-MOAT^{[C-MOAT is located in the canalicular membrane within the apical region of the hepatocyte.] [Cashore, William J. (2017). “Neonatal Bilirubin Metabolism”. Fetal and Neonatal Physiology. Elsevier. pp. 929–933. doi:10.1016/b978-0-323-35214-7.00096-2. ISBN 978-0-323-35214-7. Conjugated bilirubin is excreted into canalicular bile by way of the canalicular multispecific organic anion transport (C-MOAT) system located in the canalicular membrane within the apical region of the hepatocyte.][Koike, K; Kawabe, T; Tanaka, T; Toh, S; Uchiumi, T; Wada, M; Akiyama, S; Ono, M; Kuwano, M (1997-12-15). “A canalicular multispecific organic anion transporter (cMOAT) antisense cDNA enhances drug sensitivity in human hepatic cancer cells”. Cancer Research. 57 (24): 5475–9. ISSN 0008-5472. PMID 9407953.][Paulusma, CC; van Geer, MA; Evers, R; Heijn, M; Ottenhoff, R; Borst, P; Oude Elferink, RP (1999-03-01). “Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione”. Biochemical Journal. 338 (Pt 2): 393–401. doi:10.1042/bj3380393. PMC 1220065. PMID 10024515.][ “Diseases Associated with Hyperbilirubinemia”. library.med.utah.edu. 1995-01-05. Archived from the original on 2019-05-06.]} and MRP2^{[Zhou, J.; Tracy, T. S.; Remmel, R. P. (2010-07-28). “Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1”. Drug Metabolism and Disposition. 38 (11): 1907–1911. doi:10.1124/dmd.110.033829. ISSN 0090-9556. PMC 2967393. PMID 20668247.][Kamisako, T; Kobayashi, Y; Takeuchi, K; Ishihara, T; Higuchi, K; Tanaka, Y; Gabazza, EC; Adachi, Y (2000). “Recent advances in bilirubin metabolism research: the molecular mechanism of hepatocyte bilirubin transport and its clinical relevance”. Journal of Gastroenterology. 35 (9): 659–64. doi:10.1007/s005350070044. ISSN 0944-1174. PMID 11023036. S2CID 25491462.]} as normal human bile along with a little amount of unconjugated bilirubin as much as only 1 to 4 percent of total pigments in normal bile.^{[Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.][Erlinger, Serge; Arias, Irwin M.; Dhumeaux, Daniel (2014). “Inherited Disorders of Bilirubin Transport and Conjugation: New Insights Into Molecular Mechanisms and Consequences”. Gastroenterology. 146 (7): 1625–1638. doi:10.1053/j.gastro.2014.03.047. ISSN 0016-5085. PMID 24704527.]} That means up to 96%-99% of bilirubin in the bile are conjugated.^{[Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.][Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.]} Normally, there is just a little conjugated bilirubin escapes into the general circulation.^{[Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.]} Nonetheless, in the setting of severe liver disease, a significantly greater number of conjugated bilirubin will leak into circulation and then dissolve into the blood^{[Because conjugated bilirubin is water soluble and so does in blood.[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]]} and thereby filtered by the kidney, and only a part of the leaked conjugated bilirubin will be re-absorbed in the renal tubules, the remainder will be present in the urine making it dark-colored.^{[Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.][Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]}
  • The clinical significance of bilirubin glucuronide is involved in many conditions. Drugs that inhibit the activities of the components involved in bilirubin metabolism can give rise to accumulation of bilirubin in the blood. In comparison, conjugation of some drugs is also usually impaired if the liver cannot normally metabolize indirect bilirubin.^{[Zhou, J.; Tracy, T. S.; Remmel, R. P. (2010-07-28). “Bilirubin Glucuronidation Revisited: Proper Assay Conditions to Estimate Enzyme Kinetics with Recombinant UGT1A1”. Drug Metabolism and Disposition. 38 (11): 1907–1911. doi:10.1124/dmd.110.033829. ISSN 0090-9556. PMC 2967393. PMID 20668247.]}
    • Renal
      • When excretion of bilirubin glucuronide by the kidney is detected in the urine through urine examination, meaning that a conspicuous amount of conjugated bilirubin is present and circulating in the blood.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]}
    • Dubin–Johnson syndrome
      • In Dubin–Johnson syndrome, impaired biliary excretion of bilirubin glucuronide is due to a mutation in the canalicular multiple drug-resistance protein 2 (MRP2). A darkly pigmented liver is due to polymerized epinephrine metabolites, not bilirubin.^{[Kumar, Vinay (2007). Robbins Basic Pathology. Elsevier. p. 639.]}
    • Liver failure or hepatitis
      • If it is the liver that cannot effectively transfer the indirect bilirubin into bilirubin glucuronide and further into bilirubin di-glucuronide, the consequence will be hyperbilirubinemia or intrahepatic (or hepatocellular) jaundice. Moreover, the unconjugated hyperbilirubinemia arises in case the components of liver transfer the indirect bilirubin into bilirubin glucuronide in the rate slower than they should be.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]} This condition is associated with either decreased uptake of bilirubin into hepatocytes (Rotor syndrome^{[van de Steeg, Evita; Stránecký, Viktor; Hartmannová, Hana; Nosková, Lenka; Hřebíček, Martin; Wagenaar, Els; van Esch, Anita; de Waart, Dirk R.; Oude Elferink, Ronald P.J.; Kenworthy, Kathryn E.; Sticová, Eva; al-Edreesi, Mohammad; Knisely, A.S.; Kmoch, Stanislav; Jirsa, Milan; Schinkel, Alfred H. (2012-02-01). “Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver”. The Journal of Clinical Investigation. 122 (2): 519–528. doi:10.1172/jci59526. ISSN 0021-9738. PMC 3266790. PMID 22232210.]}) or defective intracellular protein binding. In similar fashion, the conjugated hyperbilirubinemia emerges in case the components of the liver have difficulty turning bilirubin glucuronide into bilirubin di-glucuronide.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]} Note that biliary duct blockage can also lead to conjugated hyperbilirubinemia but the pathophysiology is that backflow of bilirubin di-glucuronide with little indirect bilirubin and bilirubin glucuronide from bile duct through liver into blood plasma.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.][Weiss, Janet S.; Gautam, Anil; Lauff, John J.; Sundberg, Michael W.; Jatlow, Peter; Boyer, James L.; Seligson, David (1983-07-21). “The Clinical Importance of a Protein-Bound Fraction of Serum Bilirubin in Patients with Hyperbilirubinemia”. The New England Journal of Medicine. 309 (3): 147–150. doi:10.1056/nejm198307213090305. ISSN 0028-4793. PMID 6866015.]} These conditions are associated with either defective intracellular protein binding (for the second time) or disturbed secretion into the bile canaliculi (Dubin-Johnson syndrome^{[van de Steeg, Evita; Stránecký, Viktor; Hartmannová, Hana; Nosková, Lenka; Hřebíček, Martin; Wagenaar, Els; van Esch, Anita; de Waart, Dirk R.; Oude Elferink, Ronald P.J.; Kenworthy, Kathryn E.; Sticová, Eva; al-Edreesi, Mohammad; Knisely, A.S.; Kmoch, Stanislav; Jirsa, Milan; Schinkel, Alfred H. (2012-02-01). “Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver”. The Journal of Clinical Investigation. 122 (2): 519–528. doi:10.1172/jci59526. ISSN 0021-9738. PMC 3266790. PMID 22232210.]}). Liver failure and hepatitis are the most etiological in liver-genesis hyperbilirubinemia. In case of hyperbilirubinemia due to intrahepatic or extrahepatic bile ducts blockage, e.g. gallstone, the name is given as Post-hepatic (or obstructive) jaundice.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]} Bilirubin concentration is not a sensitive early indicator of liver diseases as the liver may have reserved its capacity in removal of bilirubin to save energy and unreserved the previously reserved capacity when encountering a sudden rise of unconjugated bilirubin. In short, there is still a chance for an ill liver to get rid of excessive unconjugated bilirubin in the blood plasma, displaying a total bilirubin level that is within normal reference range.^{[Raymond, GD; Galambos, JT (1971). “Hepatic storage and excretion of bilirubin in man”. The American Journal of Gastroenterology. 55 (2): 135–44. ISSN 0002-9270. PMID 5580257.]}
    • Crigler Najjar disease
      • In Crigler Najjar disease, there is an inherited deficiency of glucuronyl transferase resulting in high concentrations of unconjugated bilirubin appear in the plasma. Furthermore, those affected may develop kernicterus (deposits of pigment in the brain) that can cause nerve degeneration.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]}
    • Gilbert’s syndrome
      • In Gilbert’s syndrome, glucuronyl transferase activity is reduced by approximately 70%, leading to mild accumulation of unconjugated bilirubin in the plasma.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]}
    • Neonate jaundice
      • At birth, infants don’t develop enough ability to conjugate bilirubin. Up to 8% to 11% neonates will develop hyperbilirubinemia in the first week of their lives.^{[Ullah, S; Rahman, K; Hedayati, M (2016). “Hyperbilirubinemia in Neonates: Types, Causes, Clinical Examinations, Preventive Measures and Treatments: A Narrative Review Article”. Iranian Journal of Public Health. 45 (5): 558–568. PMC 4935699. PMID 27398328.][Shapiro, Steven M.; Bhutani, Vinod K.; Johnson, Lois (2006). “Hyperbilirubinemia and Kernicterus”. Clinics in Perinatology. 33 (2): 387–410. doi:10.1016/j.clp.2006.03.010. ISSN 0095-5108. PMID 16765731.]}
    • Hemolytic jaundice
      • In jaundice owing to hemolysis (Prehepatic (or hemolytic) jaundice), the pathophysiology is that overproduction of bilirubin from the extravascular or intravascular hemolysis overwhelms the capacity of the liver to excrete it. The bilirubin present in the plasma is largely unconjugated in this setting as they haven’t been taken up and conjugated by the liver.^{[Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4.]} In this case, total serum bilirubin increases while the ratio of direct bilirubin to indirect bilirubin remains 96 to 4 as up to 96%-99% of bilirubin in the bile are conjugated mentioned above.^{[Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.][Divers, Thomas J.; Barton, Michelle Henry (2018). “Disorders of the Liver”. Equine Internal Medicine. Elsevier. pp. 843–887. doi:10.1016/b978-0-323-44329-6.00013-9. ISBN 978-0-323-44329-6. then convert biliverdin to bilirubin and release it from the cell as free, insoluble bilirubin. This form of bilirubin also is referred to as indirect-reacting or unconjugated bilirubin.]}
    • Brain damage
      • See also: Kernicterus, Jaundice § Complications, and Grey matter § Clinical significance
      • Although there were some studies that showed an inverse correlation between serum bilirubin level and prevalences of ischemic coronary artery disease,^{[Breimer, LH; Wannamethee, G; Ebrahim, S; Shaper, AG (1995). “Serum bilirubin and risk of ischemic heart disease in middle-aged British men”. Clinical Chemistry. 41 (10): 1504–8. doi:10.1093/clinchem/41.10.1504. ISSN 0009-9147. PMID 7586525.]} cancer mortality,^{[Temme, EH; Zhang, J; Schouten, EG; Kesteloot, H (2001). “Serum bilirubin and 10-year mortality risk in a Belgian population”. Cancer Causes & Control. 12 (10): 887–94. doi:10.1023/A:1013794407325. ISSN 0957-5243. PMID 11808707. S2CID 19539913.]} or colorectal cancer in general population, the potential benefits of the chemopreventive function of bilirubin and their causative relations haven’t been proved.^{[ Zucker, Stephen D.; Horn, Paul S.; Sherman, Kenneth E. (2004). “Serum bilirubin levels in the U.S. population: Gender effect and inverse correlation with colorectal cancer”. Hepatology. 40 (4): 827–835. doi:10.1002/hep.20407. ISSN 0270-9139. PMID 15382174.][Namita Roy-Chowdhury; Jayanta Roy-Chowdhury. Sanjiv Chopra; Elizabeth B Rand; Shilpa Grover (eds.). “Bilirubin metabolism”. UpToDate. Archived from the original on 2017-10-24. Retrieved 2019-05-05.][excessive citations]}
        • Shapiro, Steven M. (January 2005). “Definition of the Clinical Spectrum of Kernicterus and Bilirubin-Induced Neurologic Dysfunction (BIND)”. Journal of Perinatology. 25 (1): 54–59. doi:10.1038/sj.jp.7211157. PMID 15578034. S2CID 19663259.
        • Brites, Dora (2012-05-29). “The Evolving Landscape of Neurotoxicity by Unconjugated Bilirubin: Role of Glial Cells and Inflammation”. Frontiers in Pharmacology. 3: 88. doi:10.3389/fphar.2012.00088. ISSN 1663-9812. PMC 3361682. PMID 22661946.
        • Wusthoff, Courtney J.; Loe, Irene M. (2015-01-10). “Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes”. Seminars in Fetal & Neonatal Medicine. 20 (1): 52–57. doi:10.1016/j.siny.2014.12.003. ISSN 1744-165X. PMC 4651619. PMID 25585889.
        • Radmacher, Paula G; Groves, Frank D; Owa, Joshua A; Ofovwe, Gabriel E; Amuabunos, Emmanuel A; Olusanya, Bolajoko O; Slusher, Tina M (2015-04-01). “A modified Bilirubin-induced neurologic dysfunction (BIND-M) algorithm is useful in evaluating severity of jaundice in a resource-limited setting”. BMC Pediatrics. 15 (1): 28. doi:10.1186/s12887-015-0355-2. ISSN 1471-2431. PMC 4389967. PMID 25884571.
        • Johnson, Lois; Bhutani, Vinod K. (2011). “The Clinical Syndrome of Bilirubin-Induced Neurologic Dysfunction”. Seminars in Perinatology. 35 (3): 101–113. doi:10.1053/j.semperi.2011.02.003. ISSN 0146-0005. PMID 21641482.
        • Bhutani, Vinod K.; Wong, Ronald (2015). “Bilirubin-induced neurologic dysfunction (BIND)”. Seminars in Fetal and Neonatal Medicine. 20 (1): 1. doi:10.1016/j.siny.2014.12.010. ISSN 1744-165X. PMID 25577656.
        • Press, Dove (2018-03-07). “Acute bilirubin encephalopathy and its progression to kernicterus: cur – RRN”. Research and Reports in Neonatology. 8: 33–44. doi:10.2147/RRN.S125758. Archived from the original on 2019-05-06. Retrieved 2019-05-06.
        • Smith, Margaret E.; Morton, Dion G. (2010). “Liver and Biliary System”. The Digestive System. Elsevier. pp. 85–105. doi:10.1016/b978-0-7020-3367-4.00006-2. ISBN 978-0-7020-3367-4. However, when jaundice is present it is likely that many other potentially toxic materials have also accumulated in the blood as a consequence of their reflux from the bile or impaired secretion from the hepatocyte. This can lead to impaired mental function and malaise.
        • Watchko, Jon F.; Tiribelli, Claudio (2013-11-21). Ingelfinger, Julie R. (ed.). “Bilirubin-Induced Neurologic Damage — Mechanisms and Management Approaches”. The New England Journal of Medicine. 369 (21): 2021–2030. doi:10.1056/nejmra1308124. ISSN 0028-4793. PMID 24256380.
        • Shapiro, Steven M.; Bhutani, Vinod K.; Johnson, Lois (2006). “Hyperbilirubinemia and Kernicterus”. Clinics in Perinatology. 33 (2): 387–410. doi:10.1016/j.clp.2006.03.010. ISSN 0095-5108. PMID 16765731.
  • Bilirubin diglucuronide
    • Bilirubin di-glucuronide is a conjugated form of bilirubin formed in bilirubin metabolism.^{[Chowdhury, J. R.; Chowdhury, N. R.; Wu, G.; Shouval, R.; Arias, I. M. (1981). “Bilirubin mono- and diglucuronide formation by human liver in vitro: Assay by high-pressure liquid chromatography”. Hepatology. 1 (6): 622–7. doi:10.1002/hep.1840010610. PMID 6796486.]} The hydrophilic character of bilirubin diglucuronide enables it to be water-soluble. It is pumped across the hepatic canalicular membrane into the bile by the transporter MRP2.^{[Lengyel, G.; et al. (2007-08-29). “Modulation of sinusoidal and canalicular elimination of bilirubin-glucuronides by rifampicin and other cholestatic drugs in a sandwich culture of rat hepatocytes”. Hepatology Research. Wiley. 38 (3): 300–309. doi:10.1111/j.1872-034X.2007.00255.x. PMID 17760873.]}
    • See also: Bilirubin mono-glucuronide
• Intestine, excretion in feces
  • Stercobilinogen
    • Stercobilinogen (fecal urobilinogen) is a chemical created by bacteria in the gut. It is made of broken-down hemoglobin. It is further processed to become the chemical that gives feces its brown color.^{[Stercobilinogen, drugs.com]} Bilirubin is a pigment that results from the breakdown of the heme portion of hemoglobin. The liver conjugates bilirubin, making it water-soluble; and the conjugated form is then excreted in urine as urobilinogen. Urobilinogen is colourless and is further oxidised to stercobilin which imparts colour to feces. Darkening of feces upon standing in air is due to the oxidation of residual urobilinogens to urobilins. In the intestine, bilirubin is converted by bacteria to stercobilinogen. Stercobilinogen is absorbed and excreted by either the liver or the kidney. Stercobilinogen is oxidized to stercobilin, which is responsible for the pigmentation of feces.
  • Stercobilin
    • Stercobilin is a tetrapyrrolicbile pigment and is one end-product of heme catabolism.^{[Boron W, Boulpaep E. Medical Physiology: A cellular and molecular approach, 2005. 984-986. Elsevier Saunders, United States. ISBN 1-4160-2328-3][Kay IT, Weimer M, Watson CJ (1963). “The formation in vitro of stercobilin from bilirubin” Journal of Biological Chemistry. 238:1122-3. PMID 14031566]} It is the chemical responsible for the brown color of human feces and was originally isolated from feces in 1932. Stercobilin (and related urobilin) can be used as a marker for biochemical identification of fecal pollution levels in rivers.^{[Lam, Ching-Wan; Lai, Chi-Kong; Chan, Yan-Wo (1 February 1998). “Simultaneous Fluorescence Detection of Fecal Urobilins and Porphyrins by Reversed-Phase High-Performance Thin-Layer Chromatography”. Clinical Chemistry. 44 (2): 345–346. doi:10.1093/clinchem/44.2.345. PMID 9474036.]} Stercobilin results from breakdown of the heme moiety of hemoglobin found in erythrocytes. Macrophages break down senescent erythrocytes and break the heme down into biliverdin, which rapidly reduces to free bilirubin. Bilirubin binds tightly to plasma proteins (especially albumin) in the blood stream and is transported to the liver, where it is conjugated with one or two glucuronic acid residues into bilirubin diglucuronide, and secreted into the small intestine as bile. In the small intestine, some bilirubin glucuronide is converted back to bilirubin via bacterial enzymes in the terminal ileum. This bilirubin is further converted to colorless urobilinogen. Urobilinogen that remains in the colon can either be reduced to stercobilinogen and finally oxidized to stercobilin, or it can be directly reduced to stercobilin. Stercobilin is responsible for the brown color of human feces. Stercobilin is then excreted in the feces.^{[Seyfried H, Klicpera M, Leithner C, Penner E (1976). “Bilirubin metabolism”. Wiener Klinische Wochenschrift. 88:477-82. PMID 793184]}
    • Role in disease
      • Obstructive jaundice
        • In obstructive jaundice, no bilirubin reaches the small intestine, meaning that there is no formation of stercobilinogen. The lack of stercobilin and other bile pigments causes feces to become clay-colored.^{[Seyfried H, Klicpera M, Leithner C, Penner E (1976). “Bilirubin metabolism”. Wiener Klinische Wochenschrift. 88:477-82. PMID 793184]}
      • Brown pigment gallstones
        • An analysis of two infants suffering from cholelithiasis observed that a substantial amount of stercobilin was present in brown pigment gallstones. This study suggested that brown pigment gallstones could form spontaneously in infants suffering from bacterial infections of the biliary tract.^{[ Treem, William R.; Malet, Peter F.; Gourley, Glenn R.; Hyams, Jeffrey S. (February 1989). “Bile and Stone Analysis in Two Infants With Brown Pigment Gallstones and Infected Bile”. Gastroenterology. 96 (2): 519–523. doi:10.1016/s0016-5085(89)91579-5. PMID 2642880.]}
    • Role in treatment of disease
      • A 1996 study by McPhee et al. suggested that stercobilin and other related pyrrolic pigments — including urobilin, biliverdin, and xanthobilirubic acid — has potential to function as a new class of HIV-1 protease inhibitors when delivered at low micromolar concentrations. These pigments were selected due to a similarity in shape to the successful HIV-1 protease inhibitor Merck L-700,417 (N,N-Bis(2-hydroxy-1-indanyl)-2,6-diphenylmethyl-4-hydroxy-1,7-heptandiamide). Further research is suggested to study the pharmacological efficacy of these pigments.^{[McPhee, Fiona; Caldera, Patricia S.; Bemis, Guy W.; McDonagh, Antony F.; Kuntz, Irwin D.; Craik, Charles S. (1 December 1996). “Bile pigments as HIV-1 protease inhibitors and their effects on HIV-1 viral maturation and infectivity in vitro”. Biochemical Journal. 320 (2): 681–686. doi:10.1042/bj3200681. PMC 1217983. PMID 8973584.]}
    • See also: Bile pigment, Bilirubin, Biliverdin, Heme, Urobilin
  • Urobilinogen
    • Urobilinogen is a colorless by-product of bilirubin reduction. It is formed in the intestines by bacterial action on bilirubin. About half of the urobilinogen formed is reabsorbed and taken up via the portal vein to the liver, enters circulation and is excreted by the kidney.
    • Increased amounts of bilirubin are formed in hemolysis, which generates increased urobilinogen in the gut. In liver disease (such as hepatitis), the intrahepatic urobilinogen cycle is inhibited also increasing urobilinogen levels. Urobilinogen is converted to the yellow pigmented urobilin apparent in urine.
    • The urobilinogen in the intestine is directly reduced to brownish colour stercobilin, which gives the feces their characteristic color. It can also be reduced to stercobilinogen, which can then be further oxidized to stercobilin.
    • In biliary obstruction, below-normal amounts of conjugated bilirubin reach the intestine for conversion to urobilinogen. With limited urobilinogen available for reabsorption and excretion, the amount of urobilin found in the urine is low. High amounts of the soluble conjugated bilirubin enter the circulation where they are excreted via the kidneys. These mechanisms are responsible for the dark urine and pale stools observed in biliary obstruction.
    • Low urine urobilinogen may result from complete obstructive jaundice or treatment with broad-spectrum antibiotics, which destroy the intestinal bacterial flora. (Obstruction of bilirubin passage into the gut or failure of urobilinogen production in the gut.)
    • Low urine urobilinogen levels may result from congenital enzymatic jaundice (hyperbilirubinemia syndromes) or from treatment with drugs that acidify urine, such as ammonium chloride or ascorbic acid.
    • Elevated levels may indicate hemolytic anaemia (excessive breakdown of red blood cells RBC), overburdening of the liver, increased urobilinogen production, re-absorption – a large hematoma, restricted liver function, hepatic infection, poisoning or liver cirrhosis.^{[“Urobilinogen”. Family Health Information. Retrieved 2008-03-30.][“Urobilinogen in urine”. Home test kist. Retrieved 2008-03-30.]}
    • Nomenclature
      • Urobilinogen (a.k.a. D-urobilinogen) is closely related to two other compounds: mesobilirubinogen (a.k.a. I-urobilinogen) and stercobilinogen (a.k.a. L-urobilinogen). Specifically, urobilinogen can be reduced to form mesobilirubinogen, and mesobilirubinogen can be further reduced to form stercobilinogen.^{[ “Biochemical Pathways Map No. L5 L6”. ExPASy Bioinformatics Resource Portal. Retrieved 12 December 2011.]} Confusingly, however, all three of these compounds are frequently collectively referred to as “urobilinogens”.^{[Henry’s clinical diagnosis and management by laboratory methods (PDF) (22nd ed.). Philadelphia, PA: Elsevier/Saunders. 2011. p. 543. ISBN 978-1-4377-0974-2.]}
    • Measurement
      • Urobilinogen content is determined by a reaction with Ehrlich’s reagent, which contains para-Dimethylaminobenzaldehyde and may be measured in Ehrlich units. Ehrlich’s reagent reacts with urobilinogen to give a pink-red color. One Ehrlich unit is equal to one milligram of urobilinogen per deciliter of sample (1 mg/dL).^{[Urobilinogenfrom Information and Courses MediaLab, Inc. Retrieved on Jan 8, 2009]}
• Kidney, excretion in urine
  • Urobilin
    • Urobilin or urochrome is the chemical primarily responsible for the yellow color of urine. It is a linear tetrapyrrole compound that, along with the related colorless compound urobilinogen, are degradation products of the cyclic tetrapyrrole heme. Urobilin is generated from the degradation of heme, which is first degraded through biliverdin to bilirubin. Bilirubin is then excreted as bile, which is further degraded by microbes present in the large intestine to urobilinogen. Some of this remains in the large intestine, and its conversion to stercobilin gives feces their brown color. Some is reabsorbed into the bloodstream and then delivered to kidney. When urobilinogen is exposed to air, it is oxidized to urobilin, giving urine its yellow color.^{[ John E. Hall (2016). “The liver as an organ”. Guyton and Hall Textbook of Medical Physiology, 13th edition. Elsevier. p. 885. ISBN 978-1455770052.]}
    • Importance
      • Many urine tests (urinalysis) monitor the amount of urobilin in urine, as its levels can give insight on the effectiveness of urinary tract function. Normally, urine would appear as either light yellow or colorless. A lack of water intake, for example following sleep or dehydration, reduces the water content of urine, thereby concentrating urobilin and producing a darker color of urine. Obstructive jaundice reduces biliary bilirubin excretion, which is then excreted directly from the blood stream into the urine, giving a dark-colored urine but with a paradoxically low urobilin concentration, no urobilinogen, and usually with correspondingly pale faeces. Darker urine can also be due to other chemicals, such as various ingested dietary components or drugs, porphyrins in patients with porphyria, and homogentisate in patients with alkaptonuria.
    • Voet and Voet Biochemistry Ed 3 page 1022
    • Nelson, L., David, Cox M.M., .2005. “Chapter 22- Biosynthesis of Amino Acids, Nucleotides, and Related Molecules”, pp. 856, In Lehninger Principles of Biochemistry. Freeman, New York. pp. 856
    • Bishop, Michael, Duben-Engelkirk, Janet L., and Fody, Edward P. “Chapter 19, Liver Function, Clinical Chemistry Principles, Procedures, Correlations, 2nd Ed.” Philadelphia: copyright 1992 J.B. Lippincott Company.
    • Munson-Ringsrud, Karen and Jorgenson-Linné, Jean “Urinalysis and Body Fluids, A ColorText and Atlas.” St. Louis: copyright 1995 Mosby