Chemical Education Today
Letters Photooxidation of Bilirubin to Biliverdin and Bilirubin Structure They said: Have you brought to us the truth…? (1) Two recent articles in this Journal (2, 3) dealt with bilirubin (BR), the yellow pigment of neonatal jaundice. We fully agree that BR is a fascinating biomedically-relevant tool for teaching photochemistry and stereochemistry, but also wish to point out that both articles are minefields of misinformation whose main pedagogical use might be as examples of error. “In the Laboratory” Article (2) 1. The article describes a kinetic experiment on photooxidation of BR with sunlight. It implies that the principal photoproduct is biliverdin1 (BV). Other products are not mentioned and the reaction is suggested as a possible preparative route to BV. More than half of the references in the article are questionable, and for the most part irrelevant, work from the authors’ own laboratory while more definitive earlier work (4) is ignored. BR photooxidation has been investigated extensively because of the use of phototherapy for treating neonatal jaundice (5, 6). BV was once assumed to be the main photoproduct, but later work showed that BV yields, though solvent dependent, are generally extremely low (7–9).
Figure 1. Absorption spectrum of 42 μM BR in 0.05 M NaOH superimposed on the t = 0 spectrum from Figure 2 of the supplemental material (2) (purportedly of 40 μM BR in 0.05 M NaOH).
2. Figure 1 of the article (2) and Figure 2 of the supplemental material (10) show photobleaching of BR during 20 min solar irradiation in 0.05 M NaOH. The caption to Figure 1 states that “most of the bilirubin has been converted to biliverdin”. That is clearly incorrect. The peak extinction coefficient (mM‒1 cm‒1) of BR in 0.05 M NaOH is ≤55 (11, 12)2 and those of BV are expected to be ~44 (at 381 nm) and 11 (at 720 nm), based on values in 0.1 M NaOH (12, 13). Therefore, complete conversion of BR to BV would be expected to be accompanied by replacement of the BR visible absorbance band by a slightly less intense band near 380 nm and the appearance of significant absorbance near 700 nm. The relatively weak absorbance near 380 nm and lack of absorbance near 700 nm after 20 min insolation apparent in both figures (2, 10) shows that most of the BR was not converted to BV and that little, if any, BV was formed.3 The main products are clearly colorless (4). Isolation of BV for possible use in organic synthesis, as suggested (2), would be futile. 3. The measured value of 60.05 mM‒1 cm‒1 for the absorptivity of BR at 453 nm in 0.05 M NaOH reported in the article and calculable from the first row of the “typical results” in Table 1 is not plausible. The absorptivity of BR in 0.05 M NaOH at 453 nm is ~20% lower than this (11).2 The authors compared their measured value with a literature value of 60.1 mM‒1 cm‒1. However, that value is for BR in CHCl3, which is irrelevant. We conclude that the data in Table 1 and the related kinetic plots could not have been derived from photolysis of BR in 0.05 M NaOH as described. It is also unclear why photooxidation was monitored at 453 nm since that is not the absorption maximum for BR in 0.05 M NaOH, which is close to 426–432 nm (11, 12).2 Photooxidation experiments could be done in CHCl3, but CHCl3 is unsuitable for student experiments, particularly those done using bright sunlight. 4. Figure 2 of the supplemental material (10) purports to show typical data obtained during insolation of BR in 0.05 M NaOH. However, the zero-time spectrum shown is not that of BR in 0.05 M NaOH (Figure 1 at left). It resembles, in shape and absorption maximum (~450 nm), that of BR in organic solvents. However, organic solvents were not mentioned (2, 10). 5. The authors may have “found that autoxidation can be appreciable in NaOH” (2) but that was already well-known (11, 12) and makes NaOH a poor choice of solvent. Alternative inexpensive solvents in which BR is soluble, more stable in the dark, and in which photobleaching occurs with little formation of BV are NH4OH/MeOH (MeOH containing 1% v/v concentrated aqueous NH4OH solution), dimethyl sulfoxide, or aqueous solutions of common detergents such as Triton X-100 (1%) or cetyl trimethyl ammonium bromide (10 mM) in 0.1 M phosphate buffer, pH 7.4 (9). BR undergoes photooxidation in all of these, the products consisting mainly of colorless mono and dipyrroles (4). Solvent can be dispensed with altogether, and a captivating way to demonstrate photooxidation of BR is to expose nylon or cotton fabric that has been dyed with BR to sunlight (Figure 2 below) (9).4 100-Watt desk lamps, suggested as a substitute for sunlight (10), have low emission within the BR absorption band. Fluorescent or blue LED lights might be better.
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Letters 6. The authors state that during phototherapy BR is “converted to biliverdin, which is non-toxic and is excreted” (10). In support they cite (misspelling the author’s name) a very brief out-of-date 1989 review that does not even mention biliverdin (14). There is no credible evidence that significant quantities of BV are formed or excreted during phototherapy. Even if produced, it would be rapidly reconverted to BR by BV reductase. 6. Some MSDS sheets suggest that BR and BV are toxic but we can find no clinical or scientific evidence that they are specific eye or skin irritants as stated (2).5 Except under pathological conditions, BR is not toxic.6 Toxicity data for BV is unavailable, but there is no reason to suspect either pigment of dermal or skin toxicity or to suggest they are unsafe to handle if the usual precautions for manipulating finely divided solids are taken. 7. The authors rightly state that BR is pedagogically ideal for photochemical experiments and, in the supplemental material (10), mention the clinical use of phototherapy. They are remiss in not noting that photooxidation, because of its very low quantum efficiency, plays but a small role in phototherapy and that more interesting, easily demonstrated (15), and very much faster photoisomerization reactions are crucial to the treatment’s success (5, 6, 16). “JCE Featured Molecules” Article (3) 1. The featured molecules section stressed the pedagogic importance of three-dimensional (3D) representations of molecules (3). Recognizing the 3D structures and stereochemistry of BV and BR is essential for understanding their chemical and biological properties. Unfortunately, the 3D model of BR depicted (3), and available as a manipulable version (17), is incorrect and inconsistent with the known crystal and solution structures of BR, with its lipophilicity and biochemistry, and with preferred conformations derived from molecular orbital and molecular mechanics force field calculations (5, 18–21). The most stable conformation of BR is stabilized by hydrogen bonding and has a markedly different shape from the highenergy conformer shown or from that of BV. Contrary to what was shown, each dipyrrinone chromophore in BR is almost planar and the attached propionic acid groups do not protrude into space but are intramolecularly hydrogen-bonded to amino and lactam functions. Furthermore, the structure shown on-line for BV should be described as a (Z,Z,Z) isomer not a (Z,Z) isomer, and there is presently little evidence to support the 3D structure shown for (E,E)-bilirubin. Based on the 3D structures shown online, it would be difficult to for students to understand why BR and BV have such markedly different chemical and biological properties. 2. The article showed planar constitutional structures of the (E,E) and (Z,Z) isomers of bilirubin, but inexplicably depicted the former linearly and the latter cyclically. That is liable to be particularly confusing for students. Since the point of the exercise was to show that planar representations can be misleading, it might have been clearer had both isomers been drawn in the same format, that is, either both linear or both cyclic. 3. One reference is to an article by Dinan (22). Unfortunately, that article, which aims to show students how errors arise,
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Figure 2. Photobleaching of bilirubin on cloth (9). Nylon (A) and cotton (B,C tee-shirt only) fabrics were immersed in a saturated aqueous solution of bilirubin (prepared by diluting 200 mg bilirubin, freshly dissolved in 12 mL 0.1 M NaOH, into 4000 mL 0.05 M Tris buffer, pH 8.5) for 10 min. The dyed fabrics were rinsed with buffer and distilled water, wrung, dried under vacuum, and ironed with a warm iron. Selected areas were exposed to sunlight through plateglass for several hours. Exposed areas eventually become totally bleached; covered areas remain bright yellow (dark areas in photograph). The yellow color fades only very slowly if the fabric is protected from light. A and B were photographed through a blue filter.
itself contains a major error! It suggests that photoconversion of the biosynthetic (Z,Z) isomer of BR to its (E,E) isomer, a twophoton reaction, is a significant process in phototherapy. In fact, there is little evidence to support that suggestion; formation of (Z,E)/(E,Z) isomers and other structural isomers are currently thought to be the most important processes (5, 6). 4. The confusion about structural representations was pointed out initially by us (23), not Ritter (24), as implied in the Journal and Dinan articles (2, 3, 22), and we provided the information that Ritter subsequently used in his article (24). Conclusion The articles and supplemental material on BR photooxidation (2, 3, 10) and the cited article by Dinan (22) are unsuitable didactic material except as case materials to illustrate the value of scientific scepticism and the importance of critically reading, and accurately interpreting and citing, the original literature. While BR photooxidation could form the basis for a student experiment in kinetics, the experiment would not yield data similar to that tabulated in Table 1 or spectra like those of Figure 2 of the supplemental material if done in 0.05 M NaOH as described (2, 10). Nevertheless, BR remains a biologically important, chemically interesting and pedagogically useful molecule, which students produce at a rate of about 0.25–0.5 g per day, and we encourage its use as a colorful teaching tool.
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Letters Notes 1. The blue-green pigment of avian (e.g., robin, emu) eggshells and the praying mantis. 2. The absorptivity cited for ~40 μM BR in 0.05 M NaOH is based on a single determination measured for this letter. It is somewhat higher than the published value of 46 mM‒1 cm‒1 (at 420 nm) determined in 0.1 M NaOH (11, 12). 3. Appearance of a greenish hue can be quantitatively misleading because the eye is more sensitive to green than yellow. 4. A reaction that can be used to bleach the yellow stains of fecal BR from newborn babies’ cotton diapers. 5. MSDS sheets are notoriously unreliable (See Ritter, S. K. Chem. Eng. News 2005, 83 (6), 24–26). 6. In fact, intravenous injection of BR was widely (and safely) used for many years as a liver function test and BR is a constituent of several Chinese traditional medicines.
Literature Cited
1. 2. 3. 4.
5. 6. 7. 8. 9.
10. 11.
Qur’an, 21:55. Pillay, A. E.; Salih, F. M. J. Chem. Educ. 2006, 83, 1327–1329. Coleman, W. F. J. Chem. Educ. 2006, 83, 1329. Lightner, D. A. In Bilirubin; Heirwegh, K. P. M., Brown, S. B., Eds.; CRC Press: Boca Raton, FL, 1982; Vol. I, pp 1–58. Lightner, D. A.; McDonagh, A. F. Acc. Chem. Res. 1984, 17, 417–424. McDonagh, A. F.; Lightner, D. A. Pediatrics 1985, 75, 443– 455. Lightner, D. A.; Crandall, D. C.; Gertler, S.; Quistad, G. B. FEBS Lett. 1973, 30, 309–312. McDonagh, A. F. Biochem. Biophys. Res. Comm. 1972, 48, 408–415. McDonagh, A. F.; Palma, L. A. In Chemistry and Physiology of Bile Pigments; DHEW Publication No. (NIH) 77-1100; Berk, P. D., Berlin, N. I., Eds.; U.S. Department of Health, Education, and Welfare: Bethesda, MD, 1977; pp 81–102. Pillay, A. E.; Salih, F. M. 2006. http://www.jce.divched.org/Journal/ Issues/2006/Sep/PlusSub/JCESupp/JCE2006p1327W.pdf (accessed Nov 2007). Lightner, D. A.; Cu, A.; McDonagh, A. F.; Palma, L. A. Biochem. Biophys. Res. Comm. 1976, 69, 648–657.
12. McDonagh, A. F. In The Porphyrins; Dolphin, D., Ed.; Academic Press: 1979; Vol. 6, pp 293–491. 13. McDonagh, A. F.; Palma, L. Biochem. J. 1980, 189, 193–208. 14. Epstein, J. H. In The Science of Photobiology; Smith, K. C., Ed.; Plenum Press: New York, 1989; pp 155–192. 15. McDonagh, A. F. In Photobiological Techniques; Pottier, R. H., Douglas, R. H., Mathis, P., Valenzeno, D. P., Eds.; Plenum: New York, 1991; pp 59–76. 16. Lightner, D. A.; Linnane, W. P.; Ahlfors, C. E. Pediatr. Res. 1984, 18, 696–700. 17. Coleman, W. F. 2006. http://www.jce.divched.org/JCEWWW/ Features/MonthlyMolecules/2006/Sep/index.html (accessed Nov 2007). 18. Bonnett, R.; Davies, J. E.; Hursthouse, M. B.; Sheldrick, G. M. Proc. Roy. Soc. Lond. B. 1978, 202, 249–268. 19. McDonagh, A. F. Hepatology 2002, 36, 1028–1029. 20. Nogales, D.; Lightner, D. A. J. Biol. Chem. 1995, 270, 73–77. 21. Person, R. V.; Peterson, B. R.; Lightner, D. A. J. Am. Chem. Soc. 1994, 116, 42–59. 22. Dinan, F. J. J. College Sci. Teaching 2004, 33, 18–22; http:// www3.nsta.org/main/news/stories/college_science.php?news_story_ ID=48991; http://sciencecases.org/bilirubin/bilirubin.asp (accessed Nov 2007). 23. McDonagh, A. F.; Lightner, D. A. Chem. Eng. News 2003, 81 (5), 2. 24. Ritter, S. K. Chem. Eng. News 2003, 81 (26), 29.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Feb/abs199.html Full text (HTML and PDF) Links to cited URLs and JCE articles A. F. McDonagh Division of Gastroenterology and the Liver Center University of California San Francisco San Francisco, CA 94143-0538
[email protected] D. A. Lightner Department of Chemistry, University of Nevada, Reno, Reno, NV 89557-0020
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