edited by: RON DELORENZO Middle Georgia College Cochran, Georgia 31014
Phototheraw and the Treatment of tIyperbilirubin&k A Demonstration of IWm Versus Intermolecular Hydrogen Bonding Antony C. Wilbraham
Southern Illinois University at Edwardsville Edwardsville, IL 62026
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Bilirubin is a hvdronhobic comnound and therefore not verv soluble in water. It isalsovery toxicand must be removed from the body. Bilirubin enters the rirculation and is transported to the h e r where two sugar residues, hydrophilic grvups, are attached to form the diester bilirubin dielucuronide. This diester is much more water-soluble than-bilirubin because hydrogen bonds can form between the OH groups on the sugar residues and water. ~~
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A red blood cell has a lifetime of about 120 days. Old cells
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are filtered from the blood by the spleen. The cell membranes rupture and the contents spill out. A major component of red blood cells is hemoglobin molecules. Once they are released from the cell, hemoglobin molecules undergo degradation to globin and heme. Globin, the protein portion of hemoglobin, is hydrolyzed to its individual amino acids. Heme, the nonprotein portion containing an Fez+ion, is degraded in several stages. I t begins with the removal of a single carbon from the heme ring by an enzyme-catalyzed oxidation reaction. The product of the reaction is biliuerdin, a green pigment. The iron released when heme is oxidized to biliverdin is retained in the body as a complex with the protein ferritin. I4
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6 ~ . Bilirubin digtueuronide
Reduction of a carbon-carbon double bond converts biliverdin to bilirubin, an orange-red pigment
The formation of the diester is catalyzed by an enzyme-the conjugating enzyme-which is present in mature liver cells. Bilirubin diglucuronide leaves the liver to be secreted in bile and excreted in urine and feces. Unconjugated bilirubin cannot leave the liver to be secreted in bile. Before birth, biliverdin is converted to bilirubin and crosses the placenta into the mother's liver. I t is then secreted in her bile. At birtb, an infant's liver must make the diester so that bilirubin can be secreted in bile. In order to do this its liver cells must be mature; they must contain the conjugating enzyme. Usually, the liver becomes fully functional within the first week after birth. But until it does, high levels of bilirubin accumulate in the blood (hvoerhiliruhinemia) and skin (jaundice) and cause the infa%skin to become ydlow. If the maturation period is prolonaed, bilirubin mav start to accumulate in hiain tissue. his-situation, if leftintreated, can lead to cerebral palsy, brain damage, and death. Ph~totherapy'.~ What can be done? The bilirubin molecule has extensive intramolecular hydrogen bonding-hydrogen bonding within
' Dobbs, R. H., and Cremer, R. J., "Phototherapy." Archives of
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Disease in Childhood, 50, 833 (1975). [This is interesting reading!] Lightner, D. L.. Woolridge. T. A,, and McDonagh. A. F., "Configurational isomerization of Bilirubin and the Mechanism of Jaundice Phototherapy," Biochemical and Biophysical Research Communications, 86, 235 (1977). Th s featre Presenn a collection of descr plow appl cations ano analogies desrgnea lo help sl.oents underslana some of the d ll c ~ l t concepls f r e q m l l y encomtered in Chemlrtry COnlriDYl on3 Inat wl prduce a greater appreciation and knowledge of political, religious, economic, historical, and scientificaspects of life are encouraged.
540
Journal of Chemical Education
t h e molecule-which prevents i t from forming hydrogen bonds with water. Bilirubin is therefore, very hydrophobic (water insoluble) and lipophilic (fat soluble) and accumulates in the fatty parts of the body, such as the cells of the skin. When bilirubin is exposed to white fluorescent light, or sunlight, it is converted to photobilirubin. In photobilirubin some or all of the intramolecular hydrogen bonding that was present in bilirubin is lost. So, photobilirubim can form intermolecular hydrogen bonds with water molecules making it more hydrophilic (water soluble) and less lipophilic (fat soluble) than bilirubin. Therefore, if an infant suffering from hyperbilimbinemia is exposed to fluorescent light some of the bilirubin in its skin is converted to photobilirubin. Because tbis photoproduct is more soluble in water than in fatty tissues i t leaves the skin and enters the blood circulation. From the blood i t passes to the liver and is readily secreted in bile and excreted in the urine and feces.
Compound 1 is the native isomer of biliruhin. This is itsconfiguration when it is ~roducedfrom the reduction of biliverdin. ~ & n p o u n d1has sid intramolecular hydrogen bonds (shown by dotted lines) which help confer hydrophobicity (water insolubility) and lipophilicity (fat solubility) on the molecule. Compounds 2,3, and 4 are the photoproducts which are collectively called photobilirubin. Each of these compounds has fewer intramolecular hydrogen bonds than the native isomer. They are produced when one or both of the cis configuration^ in Compound I a r t changed to trans. In thecisconfiguration the nitroeens in the vicinitv of the double bond are both on the same'side of the double-bond; in the trans configuration the nitrogens are on opposite sides of the double bond.
Structural Features
Bilirubin exists in several conformational forms as shown below.
These changes in structure lead to changes in physical properties. As intramolecular hydrogen bonding decreases, intermolecular hvdroeen .. bondine.-. with water. increases, and so photobilirubin is more hydrophilic (wate; soluhle) and less lipophilic (fat soluhle~than bilirubin.
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Demonstration Procedure Dissolve 7-8 mg of bilirubin (Sigma catalog #B-4126, cost $2.70 per 100mg) in 50 mL of chloroform. Pour 25 mL of tbis solution into
each of two 50-mL graduated cylinders fitted with ground-glass stoppers. Add 25 mL of water to each cylinder,stopper the cylinders, and wrap one completely with aluminum foil. This latter cylinder is the control. Place both cylinders in direct sunlight for 30 min and gently invert the cylinders two or three times every 5min. DO NOT SHAKE! After 30 min remove the foil from the control and conmare t.h e~contents cvlinders. ~ . of the two~~, As phothllrulrin ir produced in the uncovered cylinder it is extracted frmn the nunpolar iol\,ent,chlun,iorm, try the polar sol\,ent, water. The extraction of photobilirubin by the water produces a pale yellow aqueous layer. The aqueous layer in the covered cylinder shows no yellow coloration. ~
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Some Further lnvestigatlons To Try (1) Use a UVIvisihle spectrometer to determine the adsorption
spectrum of photobilirubin and bilirubin. (2) Expose the system to different light sources:,
(3) Determine the rate of production of photobd~ruhin.
Volume 61 Number 6 June 1984
541
