THERMAL AND PHOTOCHEMICAL REACTIONS OF BILIRUBIN I X a *
Antony F. McDonagh Departments o f Medicine and Pharmaceutical Chemistry University o f California San Francisco, California 94143
Bilirubin IX-a is the end product of heme catabolism in man and most animals. The pigment has no known physiological function and is normally excreted by the liver in a process that involves conversion to bilirubin IX-a! diglucuronide and other conjugates.' In the newborn baby the liver is functionally immature and bilirubin conjugation may be temporarily impaired shortly after birth. This causes an accumulation of bilirubin IX-a and can lead to a transient jaundice known as neonatal jaundice, physiologic jaundice of the newborn, or neonatal hyperbilirubinemia. ,2 Although neonatal jaundice is usually temporary and benign, it is not necessarily an innocuous condition, particularly when it is exacerbated by other factors such as prematurity or increased bile pigment production due to excessive hemolysis. Bilirubin IX-a! is n e u r o t ~ x i c , and ~ if the concentration of pigment in the blood becomes sufficiently high, it may diffuse into the brain and cause irreversible brain damage. Therapeutic measures for neonatal jaundice are all aimed at reducing the concentration of circulating bilirubin 1x4 and minimizing the risk of pigment entering the brain. Nowadays the most extensively used treatment is p h ~ t o t h e r a p y .In ~ phototherapy the infant is irradiated with visible light from a fluorescent light fixture, and this has the effect of bleaching the child and Despite causing a reduction in the concentration of circulating bilirubin 1x1~. the widespread use of phototherapy, the question of its safety is still somewhat c o n t r ~ v e r s i a l , and ~ * ~it has yet t o be determined how the treatment works at the molecular level. Although conjugation is the normal predominant mechanism for bilirubin IX-a! excretion, there are other less efficient and poorly characterized pathways by which the pigment can be eliminated.7 For example, in humans and animals that are genetically unable to conjugate bilirubin IX-a, although the serum level of the compound is elevated, it does not rise indefinitely and there is a steady excretion of bilirubin metabolites into the bile.7 These metabolites are thought to be degradation products, but their structures and mode of formation are presently unknown. In connection with studies on the mechanism of phototherapy and the alternate pathways of bilirubin IX-or metabolism, it became necessary to examine the stability of the pigment under light and dark conditions. In this paper the results of some of these studies are summarized and their relevance to bilirubin IX-a metabolism and photometabolism is briefly discussed. y2
* This work was aided by a grant from the United Cerebral Palsy Research and Education Foundation and by United States Public Health Service Grant AM-1 1275. 553
554
Annals New York Academy of Sciences THERMAL REACTIONS
Acid Isomerization Bilirubin IX-a rapidly isomerizes t o mixtures containing bilirubin 111-, IX-, and XIII-(r on brief treatment at room temperature with strong acids (e.g., HCl o r p-toluene sulfonic The overall reaction is shown in FIGURE 1. With weaker acids (e.g., hot acetic acid) the reaction is much slower.' The three isomers can be separated b y thin-layer chromatography (tlc) and mixtures of them can be quantitatively analyzed by preparative tlc followed by spectrophotometry.'O F o r example, when bilirubin IX-a ( 5 mg/ml in dimethyl sulfoxide) is treated for one minute with concentrated hydrochloric acid under nitrogen, the product (87% yield) is a mixture containing 23% bilirubin 111-a, 49% bilirubin IX-a, and 29% bilirubin XIII-a.' A similar mixture is obtained when an equimolar mixture of the 111- and XIII-a isomers is treated in the same way,' indicating that the reaction is reversible and reaches equilibrium rapidly. The reaction is of a type well known in polypyrrole chemistry'2*13 and has the general ionic mechanism shown in FIGURE 2. Electrophilic attack by Hf a t the a-carbon of a pyrrole ring adjacent t o the central -CH2 - bridge of bilirubin 1X-a can lead to two pairs of dipyrrylmethenes. Each of these pairs can recombine to give bilirubin IX-a, o r exchange partners t o give the other t w o isomers. For a completely random cleavage and recombination process the equilibrium mixture should contain the isomers in a 1 : 2 : 1 (111: IX: XIII) ratio. Isomer ratios obtained experimentally are close t o this, indicating a random process. However, deviations from the random ratio are observed when the reaction time is prolonged. This is due t o competing side reactions which destroy the individual isomers at different rates. Loss of the 111-a isomer is most rapid and loss of the XIII-a the least.' The acid-catalyzed isomerization of bilirubin has some preparative value for obtaining small quantities of the 111- and XIII-a isomers and can be used for the
+
M
CH3, V
CH=CH2,
P
7
CH2Cti2 C O O H
FIGURE 1. Isomerization of bilirubin IX-(u.
McDonagh : Bilirubin IX-a P
M
Bilirubin
555 P
M
18: -q Y H
(3)
(41
- wx+H P
M
P
M
X
H
H
H
H
M
f---
Y
FIGURE 2. Acid-catalyzed isomerization of bilirubin 1X-a.
small-scale preparation of novel pigments (as in the preparation of “half-naturalhalf-unnatural’’ verdins from bilirubin IX-(Y and octaethylbiladiene-a,c’ 4). The reaction could perhaps also be used for the preparation of l4C-bi1irubin (by treating bilirubin IX-a with acid in the presence of 4C-formaldehyde). Frequently, though, this reaction is a nuisance because it leads t o isomerically heterogeneous products when bilirubin IX-a is used t o prepare other bile pigments under acidic conditions. This difficulty can be circumvented, and isomer formation significantly reduced, by working under dilute conditions.‘ The acid-catalyzed isomerization of bilirubin IX-a obviously has no physiclogical significance.
Free Radical Isomerization Bilirubin 1x4 also readily isomerizes in aqueous solutions within t h e approximate pH range of 7.4-12.16 For example, when bilirubin I X a ( 8 5 pM) is warmed briefly in water at pH 8.5 in the dark, it is converted t o a randomized mixture of isomers with very little overall loss of pigment (TABLE 1). The reaction is reversible, so that the 1X-a isomer is formed when a mixture of t h e 111- and X1II-a isomers is used as starting materiaI.I6 Although the isomerization reaction occurs readily in the presence of air o r
Annals New York Academy of Sciences
556
TABLE 1 FREE RADICAL ISOMERIZATION OF BILIRUBIN IX-a* Saturating Gas Air Oxygen Argon Air Air Air Air Argon Argon Argon Air Air Air
Recovery of Bilirubin (%)
Additive None None None Ascorbic acid (570 pmol) Glutathione (330 Ltmol) Thiourea (13.1 mmol) EDTA (115 pmol) Nitric oxide t Iodine (1.7 pmol) Benzoyl peroxide$
79 44
90 91 78 36 90 33 87 13 66 12 91
0.1 M-NH3 0 0.1 M-NaOHS
Chloroform 8
Relative proportions of bilirubin isomers in product 111-0
1Xa
18 21 2 5 2 13 20 16 13 8 4 3 2
59 52 94 87 96 70 56 47 61 73 89 80 94
XIII-a 23 28 .3 8 2 17 24 37 26 20 6 16 4
* Solutions of bilirubin 1X-a ( 5 mg,8.6 pmol; isomeric purity 95%) in 0.05 M-trisbuffer, pH 8.5 (100 ml) were heated for 2 hr at 37" in the dark. Bilirubin was recovered from the solution and analyzed for isomers.16 t Reaction time, 5 min. Reaction done at 50". Solution supersaturated with benzoyl peroxide. 8 Neat solution of bilirubin I X - a ( 8 5 p M ) in designated solvent.
oxygen, it does not occur when oxygen is rigorously excluded from the solution (TABLE 1). Under aerobic conditions the reaction can be prevented by the addition of relatively high concentrations of free radical inhibitors such as ascorbic acid, glutathione, and thiourea. On the other hand, even in the absence of oxygen the isomerization can be initiated by nitric oxide, iodine, benzoyl peroxide (at 50°), o r light (see below). With these initiators, however, the reaction is not so clean and bilirubin recoveries are often low due t o oxidation t o verdinoid pigments and other competing reactions. The isomerization of bilirubin IX-a in aqueous base is therefore clearly a free radical process and its similarity t o the acid-catalyzed reaction is only superficial. The following general mechanism (where A-CH2 -B represents bilirubin IX-a)is consistent with the experimental observations. Initiation
+ A-CH2 -B AR + .CH2 -B + A-CH2-B A-CH2. + BR B-CH2 -B + A-CH2 * A-CH2 -B + *CH2-B A-CH, -B + .CH2 -A + A-CH2 - A + B-CH2 * A-CH2 * + 0 2 A - C H 2 0 0 . Re
+
Re
Propagation Termination
--f
--f
+
A-CH2
*
+ 'CH2 -B
-+
A-CH2 -CH2 -B (?)
The nature of the initiation process is unclear. However, the following observations suggest that the reaction is initiated directly by molecular oxygen rather than by tTaces of peroxides o r metal ions as in many radical reactions. (1) In the absence of added initiators, isomerization only occurs when oxygen is present. (2) The isornerization is not inhibited b y EDTA. (3) Isomerization is
(I
McDonagh : Bilirubin 1x4
557
not inhibited when the reaction is done in total darkness using very highly purified reagents. Further work is required t o elucidate the role of oxygen in the initiation reaction. The propagation step is formulated (FIGURE 3) as a bimolecular substitution reaction involving a resonance stabilized dipyrrylmethene radical as chain carrier. The substitution could be a concerted displacement reaction, but more likely involves addition followed b y homolysis, as shown. The main termination reaction probably involves trapping of dipyrrylmethene radicals by oxygen t o give peroxides, which would subsequently decompose t o dipyrrolic autoxidation products.
Bilirubin
lX -o( M
P
P
M
FIGURE 3. Radical isomerization of bilirubin IX-a. Proposed mechanism of the propagation step.
Radical isomerization of bilirubin 1x4 occurs predominantly in moderately basic aqueous solutions. In 0.1 M-NaOH o r 0.1 M-NH3 the reaction is very slow, and in chloroform it does not appear t o occur a t all (TABLE 1). This implies that a particular conformational and/or anionic form of bilirubin IX-(r is necessary for the reaction t o occur. Furthermore, the reaction does not take place when the bilirubin IX-a is bound t o albumin.16 For this reason the reaction is unlikely t o occur t o any significant extent in vivo where the pigment is mostly albumin-bound. However, it is possible that the ready formation of free radicals from unbound bilirubin 1X-a is related t o its toxicity. Albumin-bound bilirubin IX-a is nontoxic, but the free pigment can cause membrane damage17 and uncouple oxidative phosphorylation in mitochondria;' both of these effects can be caused by free r a d i c a k 2 Ready cleavage of the bilirubin molecule to radicals has not previously been noted. It remains t o be seen whether this reaction is peculiar t o rubinoid pigments o r whether it is a general reaction of dipyrrylmethanes. The radical isomerization of bilirubin IX-a is markedly concentrationdependent (TABLE 2). With an initial bilirubin 1x4 concentration of 85 /JM there is complete randomization and the pigment can be recovered in high yield. But at approximately one-fortieth this concentration there is no significant
Annals New York Academy of Sciences
558
TABLE 2 EFFECT OF DILUTION ON THE ISOMERIZATION OF BILIRUBIN IX-a IN 0.05 M-TRIS BUFFER* Initial Concentration of Bilirubin IX-a
Bilirubin Recoverv (%)
85 p M 2.1 p M
*
Relative Proportions of Bilirubin Isomers in Product (%) 111-a
IX-a
XIII-a
19
18
2s
4
59 91
23 6
pH 8.5, 31", 2 hr.
isomerization and there is a marked loss of bilirubin. Inhibition of isomerization probably occurs because dilution decreases the rate of the propagation reaction while at the same time increasing the rate of the (oxidative?) chain termination step. In addition there is a marked loss of bilirubin due t o enhanced autoxidation of the pigment at lower concentrations. Autoxidation It is well known that bilirubin IX-a is unstable in water at alkaline pHs in the presence of oxygen in the dark.23,24 With dilute solutions the autoxidation is quite rapid, as shown in FIGURE 4. The autoxidation mechanism is not known and is likely t o be complex. Unlike the previously discussed radical isomerization, autoxidation appears t o involve trace metal catalysis since it is inhibited Therefore, although the by EDTA24 o r by using ultra-pure solution^.^ autoxidation may be due in part t o the interaction of oxygen with dipyrrylmethene radical intermediates formed during bilirubin isomerization, other autoxidation reactions, not coupled t o the isomerization process, may occur. The products of bilirubin autoxidation have not been identified and characterized, b u t the loss of absorption in t h e visible region during the reaction suggests that extensive degradation of the tetrapyrrole occurs. The development of a strongly positive propentdyopent test during the reaction2 3,2 indicates that the highly water-soluble isomeric water-propentdyopent adducts (5)-(8) (FIGURE 5, R = H) are major products. Therefore, when bilirubin 1X-a is dissolved in water at moderately basic pHs in the presence of air (oxygen) t w o reactions occur, isomerization and autoxidation. The former predominates in more concentrated solutions, the latter under dilute conditions. These reactions should be taken into account during physicochemical studies on bilirubin, for example protein-binding studies, or eliminated b y working under anaerobic conditions. Both of the reactions are so that they are likely t o be inhibited if the bilirubin is bound t o albumin,' of little physiological significance under normal conditions. However, under hyperbilirubinemic conditions, as in neonatal jaundice, where a proportion of the pigment may exist in the free unbound ~ t a t e , ~ 'it, ~is ~most likely that autoxidation of bilirubin will occur. Some of t h e bilirubin derivatives7 that are excreted by jaundiced infants and animals in the absence of a functioning conjugating system are probably autoxidation products, and autoxidation may constitute one of the so-called alternate pathways of bilirubin metabolism. 6924
McDonagh : Bilirubin 1X-a
WAVELENGTH
559
(nml
FIGURE 4. Autoxidation of bilirubin IX-D in 0.05 M-tris buffer, pH 8.5. Initial bilirubin concentration, 15 pM. The solution was prepared under argon, kept for 1 hr in the dark, then bubbled with oxygen for 10 minutes and kept in an oxygen atmosphere in the dark.
M
V M
P
P
(51 M
V
M
P
171 FIGURE 5.
M M
V
Annals New York Academy of Sciences
560
PHOTOCHEMICAL REACTIONS
Pho toisom erization When deoxygenated aqueous solutions of bilirubin 1X-a are irradiated with visible light, the pigment is slowly converted t o a mixture of the 111-, IX- and XIII-a isomers (TABLE 3). In addition t h e bilirubin gradually decomposes t o uncharacterized materials. This photoisomerization reaction is the photochemical analogue of the “dark” radical isomerization already discussed, and both reactions presumably involve the same intermediates and propagation steps. In the photochemical reaction, initiating radicals could be formed by Type I photochemical p r o c e s ~ e sin~ which ~ ~ ~ ~excited triplet state bilirubin 1 x 4 abstracts a hydrogen atom from a second bilirubin molecule. TABLE 3 PHOTOISOMERIZATION OF BILIRUBIN IX-o!* Isomer Composition of Product Medium 0.05 M-tris, pH 8.5 (0.05 M-tris, pH 8.5 0.2 M-phosphate buffer, pH 8.0 chloroform 0.1 M-NaOH 64.2 rM-bovine serum albumin in 0.05 M-tris pH 8.5 Dimethyl sulfoxide
(%I
Bilirubin Recovery (%)
111-0
1X-a
61 79 59 59 64
17 2 21 7 2
61 95 54 83 96
24 10 2
36 35
3 5
94 94
3 2
*
x111-ol 22
31
-
Solutions of bilirubin IX-o! (2.5 mg; 4.3 rmol) in the tabulated media (100 ml) were irradiated under argon for 6 hr through plate glass with a 400 watt high-pressure mercury lamp. Bilirubin was recovered and analyzed by tlc-spectrophotometry. t Control, kept in the dark.
Like the dark reaction, the photochemical isomerization is very sensitive t o the medium. Almost complete isomerization occurs in water at p H 8.0 o r 8.5. In chloroform there is a small degree of photoisomerization. But irradiation of solutions of bilirubin 1X-a in 0.1 N NaOH, aqueous bovine albumin (pH 8.5), or dimethyl sulfoxide does not lead t o significant isomer formation. Prolonged irradiation of depilated, jaundiced (Gunn) rats failed t o produce detectable amounts of the 111- and XIII-a isomers in their serum. The reaction is unlikely t o be physiologically significant (this statement is not meant t o imply that other Type I radical reactions of triplet state bilirubin 1 x 4 are unlikely in vivo).
Pho toaddition Irradiation of bilirubin 1X-a in the presence of alcohols o r thiols under anaerobic conditions results in slow Markovnikoff addition of the alcohol or thiol to the exo-vinyl group of the bilirubin molecule (FIGURE 6 ) . 3 1 - 3 3 An
McDonagh : Bilirubin IX-a
Bilirubin
56 1
Ig. -o(
x = 0,s FIGURE 6. Photoaddition of alcohols and thiols to bilirubin I X a . ionic mechanism has been proposed for this reaction. Compounds which have been shown to add in this way include primary and secondary alcohols,31 2-mercaptoethan01,~~ g l ~ t a t h i o n e , ~and N-acetyl-L-cysteine. Manitto has also reported that when methanol is added to a previously irradiated solution of bilirubin in chloroform, addition of methanol t o the vinyl group still This observation is intriguing because it suggests that bilirubin IX-a may be photochemically converted to a relatively long-lived photoisomer or excited state. However, it is also possible that addition in this case may have been catalyzed by traces of acid9 produced photochemically during the initial irradiation period. Garbagnati and Manitto have suggested that conversion of bilirubin IX-a to more water-soluble and readily excretable tetrapyrroles by photoaddition of nucleophiles may be an important process in phototherapy. 34 A priori, however, it seems unlikely that photoaddition could make an important contribution to the efficacy of phototherapy because the reaction in general appears t o be rather slow and in the presence of oxygen is likely to be overwhelmed by the more rapid photooxidation of the pigment. Photooxidation
The photooxidation of bilirubin IX-a has been extensively studied because of its probable relevance to the mechanism of phototherapy. Although it has been known for a long time that the pigment is readily photooxidized, the products and mechanism of the reaction have been elucidated only recently. Photooxidation of bilirubin 1x4 in ammoniacal methanol yields biliverdin I X a ( 9 ) , methyl vinyl maleimide ( l o ) , hematinic acid imide (1 1) (FIGURE 7), and the isomeric pair of methanol-propentdyopent adducts ( 6 ) and (7) (FIGURE 5); R = CH3) as major p r o d ~ c t s . ~Traces ~ - ~ of ~ the dipyrrylmethme dialdehyde (1 2) are also formed, and a third methanol-propentdyopent isomer has recently been isolated from the photo product^.^" With water as solvent, similar products are obtained except that water-propentdyopent adducts [ ( 6 ) and (7); R = HI are formed instead of the corresponding methanol ad duct^.^^^^* The same compounds are also produced, but at a much greater rate, by sensitized photooxidation of bilirubin 1X-a in methanol or water, using rose bengal as ~ e n s i t i z e r It . ~is~ noteworthy ~~~ that all of the photoproducts that have been definitively identified, especially the propentdyopent adducts, are water-soluble and ought to be rapidly excretable if formed in vivo during phototherapy.
Annals New York Academy of Sciences
562
do
0
H
H
o&o
H
P
P P
OHC
HkL0 H
H
P=CH2CH2COon
FIGURE 7.
The products that are formed on photooxidation of bilirubin IX-a in nonhydroxylic solvents have not been well characterized. In chloroform biliverdin formation may be substantial and greater than in hydroxylic solvent^.^ 8 , 3 9 However, the yields of biliverdin are markedly concentrationdependent, and decrease with decreasing initial concentration of bilirubin I X ~ Y Gray, . ~ ~ Kulczycka, and Nicholson have shown that prolonged exposure of solutions of bilirubin in chloroform to daylight gives several products, including biliverdin, propentdyopents, maleimides, and carboxylic acids.42 Several novel tetrapyrrole pigments (rubins, rhodins, and purpurins) have been isolated in unspecified yield following photooxidation of bilirubin IX-a in pure destabilized chl~roform.~ There is now convincing evidence that bilirubin IX-a photooxidation is mainly a Type I1 p r o c e s ~involving ~ ~ ~ ~singlet ~ oxygen with the overall mechanism shown in FIGURE 8.4 Bilirubin sensitizes the formation of singlet oxygen and then reacts rapidly with it t o give products. Except perhaps for biliverdin IX-a, all of the identified photoproducts are compatible with this m e ~ h a n i s m . They ~ ~ ,apparently ~ ~ ~ ~ ~result from 1,2-addition of singlet oxygen at the enamine-like a and c bridges of the bilirubin IX-a molecule, or 1,4-addition to either of the central pair of pyrrole rings. Rearrangement or solvolysis of the resulting dioxetane or endoperoxide intermediates then yields the observed products. Biliverdin IX-a is not formed by a similar mechanism, but is the product of a separate reaction, which is probably a free-radical, Type I p r o c e s ~ . ~ It ~ .is ~interesting ' ~ ~ ~ t o note that biliverdin IX-tr is a singlet oxygen quencher and, compared t o bilirubin IX-a, is rather stable to p h o t o o x i d a t i ~ n . ~ ~ There are, therefore, a t least t w o pathways for the photooxidation of bilirubin IX-a.One, a Type I1 process involving singlet oxygen, leads directly t o mono- and dipyrroles without involving biliverdin IX-a as an intermediate. The other, probably a Type I reaction, leads initially t o biliverdin IX-a and then
*
344
McDonagh : Bilirubin IX-a
hv
Bi 1 i rubin 1 3
(Bi irubin)* *
b
1
563
* (Bilirubin)
*
3
P (Bilirubin)
3
(Bi irubin) + O2
1 Bi irubin + 0
2
-
______)
1
Bilirubin + O2 Products
FIGURE 8. Singlet oxygen mechanism for bilirubin IX-(u photooxidation.
slowly t o further degradation products. The relative importance of these t w o pathways will vary according to the experimental conditions, particularly solvent, oxygen tension and bilirubin IX-a concentration. This may account for the apparently ’conflicting reports regarding the amount of biliverdin produced on photooxidation of bilirubin (for example, see Reference 46). However, in general, in the presence of air the Type I1 pathway appears t o predominate. Of the two photooxidation pathways, the radical process leading to biliverdin is likely t o be of little significance in phototherapy since biliverdin can be readily ’ radical converted back again to bilirubin enzymatically in v ~ v o . ~Nevertheless, reactions leading to products other than biliverdin could operate during phototherapy. In this regard it should be noted that binding of bilirubin IX-a t o albumin is likely to increase the probability of a Type I mechanism.48 Photooxidation of bilirubin IX-a by the singlet oxygen pathway in vivo seems highly likely, particularly in the skin and peripheral fatty tissues. However, this has not yet been directly demonstrated. Since bilirubin 1x4 is a photosensitizer, one potential risk of phototherapy is photodynamic damage. However, compared to porphyrins and other well known singlet oxygen sensitizers, bilirubin IX-a is a poor photosensitizer. In addition, it reacts very rapidly with singlet oxygen (*lop9 M-’ sec-1);49,50 more rapidly in fact than any other compound yet studied. This combination of properties may well protect the jaundiced child from significant singlet oxygen-induced photodynamic injury during treatment. Formation of singlet oxygen is not likely to be excessive, and any that is formed will tend t o be rapidly scavenged by the bilirubin, which will itself be destroyed in the process.
PHOTOMETABOLISM OF BILIRUBIN I X - ~ l Although much is known about the photochemistry of bilirubin IX-a in vifro, relatively little is known concerning the photochemistry of the pigment in the intact animal. Irradiation of rats or infants with unconjugated hyperbilirubinemia causes a fall in the concentration of the pigment in the serum, but the chemistry underlying this phenomenon is not well understood. Now that phototherapy is so widely used for treating neonatal jaundice, discovering how it works has become a matter of some urgency. Until the mechanism is known it
Annals New York Academy of Sciences
564
will be difficult t o fully assess the safety of the treatment o r lay down effective guidelines for its use. Much of what is known about the mechanism of phototherapy results from studies o n Gunn rats. These are a mutant strain of Wistar rats that have a genetic Homozygous Gunn rats lack a functiondefect in bilirubin ing bilirubin glucuronyl transferase enzyme system and are unable t o conjugate and excrete the pigment in the normal way. Consequently, they have a lifelong unconjugated hyperbilirubinemia and are considered t o be a good model for the human neonate with transient unconjugated hyperbilirubinemia o r permanent unconjugated hyperbilirubinemia (Crigler-Najjar syndrome). Experiments on Gunn ratss3 and jaundiced infantss4 labeled with I4C-bilirubin have shown that there is an enhancement in the excretion of radioactive label when the subject is exposed to visible light. Of the total amount of label excreted, the greatest proportion is into the bile, and, as first shown by O ~ t r o w , ~the effect of, phototherapy on biliary excretion is particularly striking. FIGURE 9 shows the changes in the excretion of radioactivity by a labeled Gunn rat during a light-dark-light cycle. The method was essentially the same as O s t r o w ’ ~ .Prior ~ ~ t o the experiment the rat was shaved dorsally and injected with a pulse of biosynthetic l4C-bilirubin IX-a26 and then left in the dark for about twelve hours to allow exogenous labeled pigment to equilibrate with endogenous material. Bile was collected continuously throughout the experiment via a polythene tube inserted into the bile duct and fractions were taken every four hours for counting. During the “light” period the creature, with head covered, was irradiated with visible light from a fluorescent light fixture (Duro-Test Vitalite). Even in the dark there is some excretion of bilirubin-derived radioactivity. This is not due t o excretion of bilirubin IX-a itself, but to unidentified
4
I
LIGHT
j
I
E:
? P
u
10
HOURS AFTER
“C
INJECTION
FIGURE 9. Total biiiary excretion of 14C by a male hornozygous Gunn rat during a dark-light-dark cycle. Light was from Vitalite fluorescent tubes delivering an illumination of 500 foot-candles at the rat’s back.
McDonagh : Bilirubin IXiv
565
derivativest which, as discussed earlier, may include water-soluble autoxidation products. immediately following irradiation there is a very pronounced increase in isotope excretion, which gradually abates after the light is extinguished. Significantly, throughout the entire experiment the bile flow remained essentially constant, indicating that the photobiological response reflects an increased output of isotope and not merely an increase in the biliary concentration of the isotope. As originally shown by O s t r o ~ , ~ the light-induced increment in the amount of radioactive material excreted is made up of t w o components. One component contains unidentified bilirubin derivatives and the other consists of unconjugated bulirubin IX-a. The bilirubin component is substantial5 and may account for u p t o 60% of the photostimulated excretion of radioactivity. Recent experiments suggest that phototherapy causes qualitatively similar effects in jaundiced ~ h i l d r e n . ~ Phototherapy, therefore, is effective in reducing serum bilirubin levels because it causes biliary excretion of the pigment itself and compounds derived from it. At present, it is a mystery how light stimulates the excretion of unconjugated bilirubin; possibly this is not due t o a direct photochemical reaction at all. Since none of the other derivatives that accompany the bilirubin have been identified, their origin, too, remains obscure. However, it is likely that these compounds are derived, a t least in part, from photooxidation of bilirubin in t h e skin and extravascular tissue. As previously pointed out, bilirubin IX-a is a rather poor photosensitizer. In vitro, its photooxidation is accelerated by the addition of better singlet oxygen sensitizers such as rose bengal, methylene blue, or p ~ r h y r i n s . ~ These observations suggested that by using photosensitizers in vivo it might be possible to magnify the effect of light in reducing serum bilirubin concentrations. FIGURE 10 shows the effect of intravenously administered “hematoporphyrin” ,4495
Serum
Bilirubin (%A] 30
It1
20 10 0 10 20
30
(-1
40
----- wiinoui
50
-
60
HEHATOPORPHYRIN
AFTER HEMATOPORPHYRIN
70
80
TIME (hrr)
FIGURE 10. Effect of hematoporphyrin on the efficacy of phototherapy in a Gunn rat. The rat was injected (i.v.) with saline solution and subsequently hematoporphyrin (0.5 mg/100 g) at t = 0. After 1 hr the rat was irradiated (Vitalite) for 5.5 hr and then kept in the dark for > 65 hr. Serum bilirubin values were determined by the diazo method on tail-vein blood samples and are expressed as a percentage of change from the serum bilirubin concentration at the start of the experiment.
t See Reference 43 for evidence regarding the structure and origin of some of these derivatives.
566
Annals New York Academy of Sciences
on the efficacy of phototherapy in a Gunn rat.$ Brief irradiation of the animal (dorsally shaved) with a Vitalite fixture produced only a modest decrease in the serum bilirubin concentration, whereas a similar period of irradiation following injection of the animal with “hematoporphyrin” resulted in a pronounced drop in the serum bilirubin level (FIGURE 10). Hematoporphyrin had no effect on serum bilirubin levels when the animal was kept in the dark. A similar effect has been noted b y Kostenbauder and Sanvordeker using exogenously administered riboflavin as photosensitizer. These experiments d o not prove that a singlet oxygen Type I 1 mechanism is involved in phototherapy, although they lend credence t o the possibility. However, they d o suggest that endogenous photosensitizers such as porphyrins or riboflavin might play some part in causing the dissappearance of bilirubin 1X-cy during phototherapy. SUMMARY
In solution in the dark bilirubin IX-(r readily undergoes autoxidation and isomerization. The latter can occur by an ionic or a free radical process, depending on the conditions. Irradiation of bilirubin IX-a in vitro can lead to photoisomerization, photoaddition of alcohols and thiols, o r photooxidation. Photooxidation can proceed by two pathways: one is a selfsensitized singlet oxygen process leading rapidly to mono- and dipyrroles, and the other leads initially t o biliverdin and may involve a free radical mechanism. lrradiation of jaundiced rats causes biliary excretion of the pigment and enhances the excretion of bilirubin derivatives. The overall effect is t o lower the concentration of bilirubin in the serum. This effect can be enhanced by administration of appropriate photosensitizers.
ACKNOWLEDGMENTS
I wish t o thank Mr. Francis K. Assisi and Mrs. Lucita A. Palma for their technical assistance, and Professor R. Schmid for his cooperation and support. This work was aided by generous support and equipment given b y the Duro-Test Corporation, North Bergen, New Jersey. REFERENCES 1. SCHMID, R. 1972. Hyperbilirubinemia. In The Metabolic Basis of Inherited Disease, 3rd Ed. J. B. Stanbury, J. B. Wyngaarden & D. S. Frederickson, Eds. 1141-1178. McGraw-Hill, Inc. New York, N.Y. 2. THALER, M. M. 1972. Neonatal hyperbilirubinemia. Seminars Hematol. 9 ( 2 ) : 107-112. 3. DIAMOND, I. 1969. Bilirubin binding and kernicterus. Advan. Pediat. 16: 99-1 19. 4. LUCEY. J. F. 1972. Neonatal phototherapy: uses, problems and questions. Seminars Hematol. 9 (2): 127-135. $ The hematoporphyrin used was commercial material treated with acetic acid-sulfuric acid according to the method of Lipson and Balde~.~’Subsequent work has shown this material to be a mixture of porphyrins containing hematoporphyrin as a major constituent.
McDonagh : Bilirubin I X a
567
5. SCHMID, R. 1971. More light on neonatal hyperbilirubinemia. New Eng. J. Med. 285: 520-522. 6. Editorial. 1972. Phototherapy in neonatal jaudice. Brit. Med. J. 2: 62-63. 7. SCHMID, R. & L. HAMMAKER. 1963. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 42: 1720-1734. 8. MCDONAGH, A. F. & F. ASSISI. 1972. Direct evidence for the acid-catalyzed isomeric scrambling of bilirubin IX-a. J. Chem. SOC.Chem. Commun.: 117-119. 9. MANITTO, P. & D. MONTI. 1973. Acid-catalyzed addition of alcohols and thiols t o bilirubin. Experientia 29: 137-139. 10. MCDONAGH, A. F. & F. ASSISI. 1971. Commercial bilirubin: A trinity of isomers. FEBS Lett. 18: 315-317. 11. MCDONAGH, A. F. & F. ASSISI. Unpublished observations. 12. MAUZERALL, D. 1960. The thermodynamic stability of porphyrinogens. J. Amer. Chem. SOC.82: 2601 -2605. 13. JACKSON, A. H., G. W. KENNER & J. WASS. 1972. Pyrroles and related compounds. Part XX. Synthesis of Coproporphyrins. J. Chem. SOC. Perkin Trans. 1.: 1475-1483. 14. BONNETT, R. & A. F. MCDONAGH. 1970. The isomeric heterogeneity of biliverdin dimethyl ester derived from bilirubin. J. Chem. SOC.,D: 238-239. 15. BONNETT, R. & A. F. MCDONAGH. Unpublished observations. 16. MCDONAGH, A. F. & F. ASSISI. 1972. The ready isomerization of bilirubin IX-or in aqueous solution. Biochem. J. 129: 797-800. 17. CHEUNG, W. H., A. SAWITSKY & H. D. ISENBERG. 1966. The effect of bilirubin on the mammalian erythrocyte. Transfusion 6: 475-486. 18. ZETTERSTROM, R. & L. ERNSTER. 1956. Bilirubin, an uncoupler of oxidative phosphorylation in isolated mitochondria. Nature 178: 1335-1337. 19. VOGT, M. T. & R. E. BASFORD. 1968. The effect of bilirubin on the energy metabolism of brain mitochondria. J. Neurochem. 15: 1313-1320. 20. MUSTAFA, M. G., M. L. COWGER & T. E. KING. 1969. Effects of bilirubin on mitochondria1 reactions. J. Biol. Chem. 244: 6403-6414. 21. TAPPEL, A. L. 1973. Lipid peroxidation damage t o cell components. Fed. Proc. 32: 1870-1874. 22. COLEMAN, P. S. 1973. Uncoupling of oxidative phosphorylation by a stable free radical and its diamagnetic homolog. Riochim. Biophys. Acta 305: 179-184. 23. BINGOLD, K. 1935. Weitere untersuchungen zur formulierung eines biologischchemischen blutkreislaufes. Klin. Wochenschr. 14: 1287- 1289. 24. FOG, J. & B. BUGGE-ASPERHEIM. 1964. Stability of bilirubin. Nature 203: 756-757. 25. MCDONAGH, A. F. & L. PALMA. Unpublished observations. 26. OSTROW, J. D., L. HAMMAKER & R. SCHMID. 1961. The preparation of crystalline bilirubin-C14. J. Clin. Invest. 40: 1442-1452. 27. ODELL, G . B. 1959. The dissociation of bilirubin from albumin and its clinical implications. J. Pediat. 55: 268--279. 28. NAKAMURA, H. & R. LARDINOIS. 1972. Unbound bilirubin in icteric newborns. Biol. Neonate 21: 400-417. 29. SCHENCK, G . 0. 1963. Photosensitization. Ind. Eng. Chem. 55: 40-43. 30. GOLLNICK, K. 1968. Type 11 photooxygenation reactions in solution. Advan. Photochem. 6: 1-22. 31. MANITTO, P. 1971. Photochemistry of bilirubin. Experientia 27: 1147-1 149. 32. MANITTO, P. & D. MONTI. 1972. Photoaddition of sulphydryl groups t o bilirubin in vitro. Experientia 28: 379-380. 33. MANITTO, P., D. MONTI & E. GARBAGNATI. 1972. Photochemical addition of N-acetyl-L-cysteine and glutathione to bilirubin in vitro and its relevance to phototherapy of jaundice. Farmaco, Ed. Sci. 27: 999-1002. 34. GARBAGNATI, E. & P. MANITTO. 1973. A new class of bilirubin photoderivatives obtained in vitro and their possible formation in jaundiced infants. J. Pediat. 83: 109-1 15.
568
Annals New York Academy of Sciences
35. LIGHTNER, D. A. & G. B. QUISTAD. 1972. Methylvinylmaleimide from bilirubin photooxidation. Science 175: 324. 36, LIGHTNER, D. A. & G. B. QUISTAD. 1972. Hematinic acid and propentdyopents from bilirubin photooxidation in vitro. FEBS Lett. 25: 94-96. 37. LIGHTNER, D. A. & G. B. QUISTAD. 1972. h i d e products from photo-oxidation of bilirubin and mesobilirubin. Nature New Biol. 236: 203 -205. 38. BONNETT, R. & J. C. M. STEWART. 1972. Photo-oxidation of bilirubin in hydroxylic solvents: propentdyopent adducts as major products. J. Chem. SOC. Chem. Commun.: 596. 39. LIGHTNER, D. A., D. C. CRANDALL, S. GERTLER & G. B. QUISTAD. 1973. On the formation of biliverdin during photooxygenation of bilirubin in vitro. FEBS Lett. 30: 309-312. 40. BONNETT, R. & J. C. M. STEWART. Personal communication. 41. BONNETT, R. & J. C. M. STEWART. 1972. Singlet oxygen in the photo-oxidation of bilirubin in hydroxylic solvents. Biochem. J. 130: 895-897. 42. GRAY, C. H., A. KULCZYCKA & D. C. NICHOLSON. 1972. The photodecomposition of bilirubin and other bile pigments. J. Chem. Soc. Perkin Trans. I.: 28 8 -294. 43. BERRY, C. S., J. E. ZAREMBO & J. D. OSTROW. 1972. Evidence for conversion of bilirubin to dihydroxyl derivatives in the Gunn rat. Biochem. Biophys. Res. Commun. 49: 1366-1375. 44. MCDONAGH, A. F. 1971. The role of singlet oxygen in bilirubin photo-oxidation. Biochem. Bioph.ys. Res. Commun. 44: 1306-1311. 45. MCDONAGH, A. F. 1972. Evidence for singlet oxygen quenching by biliverdin I X a dimethyl ester and its relevance to bilirubin photo-oxidation. Biochem. Biophys. Res. Commun. 48: 408-415. 46. DAVIES, R. E. & S. J. KEOHANE. 1970. Some aspects of the photochemistry of bilirubin. Boll. Chim. Farm. 109: 589-598. 47. TENHUNEN, R., M. E. ROSS, H. S. MARVER & R. SCHMID. 1970. Reduced nicotinamide-adenine dinucbotide phosphate dependent biliverdin reductase: partial purification and characterization. Biochemistry 9: 298-303. 48. BELLIN, J. S. 1968. Photophysical and photochemical effects of dye binding. Photochem. Photobiol. 8: 383-392. 49. LEE, J., 1. B. C. MATHESON, J. E. WAMPLER, R. D. ETHERIDGE & N. U. CURRY. 1973. Inhibition of laser generated singlet oxygen reaction with bilirubin by superoxide dismutase. Fed. Proc. 32: 661 (abstract). 50. FOOTE, C. S. Personal communication. 51. GUNN, C. H. 1938. Hereditary acholuric jaundice in a new mutant strain of rats. J. Heredity 29: 137-139. 52. SCHMID, R., J. AXELROD, L. HAMMAKER & R. L. SWARM. 1958. Congenital jaundice in rats due to a defect in glucuronide formation. J. Clin. Invest. 37: 1123-1130. 53. OSTROW, J. D. 1971. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 50: 707-718. 54. CALLAHAN, E. W., M. M. THALER, M. KARON, K. BAUER & R. SCHMID. 1970. Phototherapy of severe unconjugated hyperbilirubinemia: formation and removal of labeled bilirubin derivatives. Pediatrics 46: 841 -848. 55. LUND, H. T. & J. JACOBSEN. 1972. Influence of phototherapy on unconjugated bilirubin in duodenal bile of newborn infants with hyperbilirubinemia. Acta Paediat. Scand. 61: 693-696. 56. CREMER, R. J., P. W. PERRYMAN & D. H. RICHARDS. 1958. Influence of light on the hyperbilirubinaemia of infants. Lancet 1: 1094- 1097. 57. LIPSON, R. L. & E. J. BALDES. 1960. The photodynamic properties of a particular hematoporphyrin derivative. Arch. Dermatol. 82: 508-5 16. 58. KOSTENBAUDER, H. B. & D. R. SANVORDEKER. 1972. Riboflavin enhancement of bilirubin catabolism in vivo. Experientia 29: 282-283.
Antony F. McDonagh Departments o f Medicine and Pharmaceutical Chemistry University o f California San Francisco, California 94143
Bilirubin IX-a is the end product of heme catabolism in man and most animals. The pigment has no known physiological function and is normally excreted by the liver in a process that involves conversion to bilirubin IX-a! diglucuronide and other conjugates.' In the newborn baby the liver is functionally immature and bilirubin conjugation may be temporarily impaired shortly after birth. This causes an accumulation of bilirubin IX-a and can lead to a transient jaundice known as neonatal jaundice, physiologic jaundice of the newborn, or neonatal hyperbilirubinemia. ,2 Although neonatal jaundice is usually temporary and benign, it is not necessarily an innocuous condition, particularly when it is exacerbated by other factors such as prematurity or increased bile pigment production due to excessive hemolysis. Bilirubin IX-a! is n e u r o t ~ x i c , and ~ if the concentration of pigment in the blood becomes sufficiently high, it may diffuse into the brain and cause irreversible brain damage. Therapeutic measures for neonatal jaundice are all aimed at reducing the concentration of circulating bilirubin 1x4 and minimizing the risk of pigment entering the brain. Nowadays the most extensively used treatment is p h ~ t o t h e r a p y .In ~ phototherapy the infant is irradiated with visible light from a fluorescent light fixture, and this has the effect of bleaching the child and Despite causing a reduction in the concentration of circulating bilirubin 1x1~. the widespread use of phototherapy, the question of its safety is still somewhat c o n t r ~ v e r s i a l , and ~ * ~it has yet t o be determined how the treatment works at the molecular level. Although conjugation is the normal predominant mechanism for bilirubin IX-a! excretion, there are other less efficient and poorly characterized pathways by which the pigment can be eliminated.7 For example, in humans and animals that are genetically unable to conjugate bilirubin IX-a, although the serum level of the compound is elevated, it does not rise indefinitely and there is a steady excretion of bilirubin metabolites into the bile.7 These metabolites are thought to be degradation products, but their structures and mode of formation are presently unknown. In connection with studies on the mechanism of phototherapy and the alternate pathways of bilirubin IX-or metabolism, it became necessary to examine the stability of the pigment under light and dark conditions. In this paper the results of some of these studies are summarized and their relevance to bilirubin IX-a metabolism and photometabolism is briefly discussed. y2
* This work was aided by a grant from the United Cerebral Palsy Research and Education Foundation and by United States Public Health Service Grant AM-1 1275. 553
554
Annals New York Academy of Sciences THERMAL REACTIONS
Acid Isomerization Bilirubin IX-a rapidly isomerizes t o mixtures containing bilirubin 111-, IX-, and XIII-(r on brief treatment at room temperature with strong acids (e.g., HCl o r p-toluene sulfonic The overall reaction is shown in FIGURE 1. With weaker acids (e.g., hot acetic acid) the reaction is much slower.' The three isomers can be separated b y thin-layer chromatography (tlc) and mixtures of them can be quantitatively analyzed by preparative tlc followed by spectrophotometry.'O F o r example, when bilirubin IX-a ( 5 mg/ml in dimethyl sulfoxide) is treated for one minute with concentrated hydrochloric acid under nitrogen, the product (87% yield) is a mixture containing 23% bilirubin 111-a, 49% bilirubin IX-a, and 29% bilirubin XIII-a.' A similar mixture is obtained when an equimolar mixture of the 111- and XIII-a isomers is treated in the same way,' indicating that the reaction is reversible and reaches equilibrium rapidly. The reaction is of a type well known in polypyrrole chemistry'2*13 and has the general ionic mechanism shown in FIGURE 2. Electrophilic attack by Hf a t the a-carbon of a pyrrole ring adjacent t o the central -CH2 - bridge of bilirubin 1X-a can lead to two pairs of dipyrrylmethenes. Each of these pairs can recombine to give bilirubin IX-a, o r exchange partners t o give the other t w o isomers. For a completely random cleavage and recombination process the equilibrium mixture should contain the isomers in a 1 : 2 : 1 (111: IX: XIII) ratio. Isomer ratios obtained experimentally are close t o this, indicating a random process. However, deviations from the random ratio are observed when the reaction time is prolonged. This is due t o competing side reactions which destroy the individual isomers at different rates. Loss of the 111-a isomer is most rapid and loss of the XIII-a the least.' The acid-catalyzed isomerization of bilirubin has some preparative value for obtaining small quantities of the 111- and XIII-a isomers and can be used for the
+
M
CH3, V
CH=CH2,
P
7
CH2Cti2 C O O H
FIGURE 1. Isomerization of bilirubin IX-(u.
McDonagh : Bilirubin IX-a P
M
Bilirubin
555 P
M
18: -q Y H
(3)
(41
- wx+H P
M
P
M
X
H
H
H
H
M
f---
Y
FIGURE 2. Acid-catalyzed isomerization of bilirubin 1X-a.
small-scale preparation of novel pigments (as in the preparation of “half-naturalhalf-unnatural’’ verdins from bilirubin IX-(Y and octaethylbiladiene-a,c’ 4). The reaction could perhaps also be used for the preparation of l4C-bi1irubin (by treating bilirubin IX-a with acid in the presence of 4C-formaldehyde). Frequently, though, this reaction is a nuisance because it leads t o isomerically heterogeneous products when bilirubin IX-a is used t o prepare other bile pigments under acidic conditions. This difficulty can be circumvented, and isomer formation significantly reduced, by working under dilute conditions.‘ The acid-catalyzed isomerization of bilirubin IX-a obviously has no physiclogical significance.
Free Radical Isomerization Bilirubin 1x4 also readily isomerizes in aqueous solutions within t h e approximate pH range of 7.4-12.16 For example, when bilirubin I X a ( 8 5 pM) is warmed briefly in water at pH 8.5 in the dark, it is converted t o a randomized mixture of isomers with very little overall loss of pigment (TABLE 1). The reaction is reversible, so that the 1X-a isomer is formed when a mixture of t h e 111- and X1II-a isomers is used as starting materiaI.I6 Although the isomerization reaction occurs readily in the presence of air o r
Annals New York Academy of Sciences
556
TABLE 1 FREE RADICAL ISOMERIZATION OF BILIRUBIN IX-a* Saturating Gas Air Oxygen Argon Air Air Air Air Argon Argon Argon Air Air Air
Recovery of Bilirubin (%)
Additive None None None Ascorbic acid (570 pmol) Glutathione (330 Ltmol) Thiourea (13.1 mmol) EDTA (115 pmol) Nitric oxide t Iodine (1.7 pmol) Benzoyl peroxide$
79 44
90 91 78 36 90 33 87 13 66 12 91
0.1 M-NH3 0 0.1 M-NaOHS
Chloroform 8
Relative proportions of bilirubin isomers in product 111-0
1Xa
18 21 2 5 2 13 20 16 13 8 4 3 2
59 52 94 87 96 70 56 47 61 73 89 80 94
XIII-a 23 28 .3 8 2 17 24 37 26 20 6 16 4
* Solutions of bilirubin 1X-a ( 5 mg,8.6 pmol; isomeric purity 95%) in 0.05 M-trisbuffer, pH 8.5 (100 ml) were heated for 2 hr at 37" in the dark. Bilirubin was recovered from the solution and analyzed for isomers.16 t Reaction time, 5 min. Reaction done at 50". Solution supersaturated with benzoyl peroxide. 8 Neat solution of bilirubin I X - a ( 8 5 p M ) in designated solvent.
oxygen, it does not occur when oxygen is rigorously excluded from the solution (TABLE 1). Under aerobic conditions the reaction can be prevented by the addition of relatively high concentrations of free radical inhibitors such as ascorbic acid, glutathione, and thiourea. On the other hand, even in the absence of oxygen the isomerization can be initiated by nitric oxide, iodine, benzoyl peroxide (at 50°), o r light (see below). With these initiators, however, the reaction is not so clean and bilirubin recoveries are often low due t o oxidation t o verdinoid pigments and other competing reactions. The isomerization of bilirubin IX-a in aqueous base is therefore clearly a free radical process and its similarity t o the acid-catalyzed reaction is only superficial. The following general mechanism (where A-CH2 -B represents bilirubin IX-a)is consistent with the experimental observations. Initiation
+ A-CH2 -B AR + .CH2 -B + A-CH2-B A-CH2. + BR B-CH2 -B + A-CH2 * A-CH2 -B + *CH2-B A-CH, -B + .CH2 -A + A-CH2 - A + B-CH2 * A-CH2 * + 0 2 A - C H 2 0 0 . Re
+
Re
Propagation Termination
--f
--f
+
A-CH2
*
+ 'CH2 -B
-+
A-CH2 -CH2 -B (?)
The nature of the initiation process is unclear. However, the following observations suggest that the reaction is initiated directly by molecular oxygen rather than by tTaces of peroxides o r metal ions as in many radical reactions. (1) In the absence of added initiators, isomerization only occurs when oxygen is present. (2) The isornerization is not inhibited b y EDTA. (3) Isomerization is
(I
McDonagh : Bilirubin 1x4
557
not inhibited when the reaction is done in total darkness using very highly purified reagents. Further work is required t o elucidate the role of oxygen in the initiation reaction. The propagation step is formulated (FIGURE 3) as a bimolecular substitution reaction involving a resonance stabilized dipyrrylmethene radical as chain carrier. The substitution could be a concerted displacement reaction, but more likely involves addition followed b y homolysis, as shown. The main termination reaction probably involves trapping of dipyrrylmethene radicals by oxygen t o give peroxides, which would subsequently decompose t o dipyrrolic autoxidation products.
Bilirubin
lX -o( M
P
P
M
FIGURE 3. Radical isomerization of bilirubin IX-a. Proposed mechanism of the propagation step.
Radical isomerization of bilirubin 1x4 occurs predominantly in moderately basic aqueous solutions. In 0.1 M-NaOH o r 0.1 M-NH3 the reaction is very slow, and in chloroform it does not appear t o occur a t all (TABLE 1). This implies that a particular conformational and/or anionic form of bilirubin IX-(r is necessary for the reaction t o occur. Furthermore, the reaction does not take place when the bilirubin IX-a is bound t o albumin.16 For this reason the reaction is unlikely t o occur t o any significant extent in vivo where the pigment is mostly albumin-bound. However, it is possible that the ready formation of free radicals from unbound bilirubin 1X-a is related t o its toxicity. Albumin-bound bilirubin IX-a is nontoxic, but the free pigment can cause membrane damage17 and uncouple oxidative phosphorylation in mitochondria;' both of these effects can be caused by free r a d i c a k 2 Ready cleavage of the bilirubin molecule to radicals has not previously been noted. It remains t o be seen whether this reaction is peculiar t o rubinoid pigments o r whether it is a general reaction of dipyrrylmethanes. The radical isomerization of bilirubin IX-a is markedly concentrationdependent (TABLE 2). With an initial bilirubin 1x4 concentration of 85 /JM there is complete randomization and the pigment can be recovered in high yield. But at approximately one-fortieth this concentration there is no significant
Annals New York Academy of Sciences
558
TABLE 2 EFFECT OF DILUTION ON THE ISOMERIZATION OF BILIRUBIN IX-a IN 0.05 M-TRIS BUFFER* Initial Concentration of Bilirubin IX-a
Bilirubin Recoverv (%)
85 p M 2.1 p M
*
Relative Proportions of Bilirubin Isomers in Product (%) 111-a
IX-a
XIII-a
19
18
2s
4
59 91
23 6
pH 8.5, 31", 2 hr.
isomerization and there is a marked loss of bilirubin. Inhibition of isomerization probably occurs because dilution decreases the rate of the propagation reaction while at the same time increasing the rate of the (oxidative?) chain termination step. In addition there is a marked loss of bilirubin due t o enhanced autoxidation of the pigment at lower concentrations. Autoxidation It is well known that bilirubin IX-a is unstable in water at alkaline pHs in the presence of oxygen in the dark.23,24 With dilute solutions the autoxidation is quite rapid, as shown in FIGURE 4. The autoxidation mechanism is not known and is likely t o be complex. Unlike the previously discussed radical isomerization, autoxidation appears t o involve trace metal catalysis since it is inhibited Therefore, although the by EDTA24 o r by using ultra-pure solution^.^ autoxidation may be due in part t o the interaction of oxygen with dipyrrylmethene radical intermediates formed during bilirubin isomerization, other autoxidation reactions, not coupled t o the isomerization process, may occur. The products of bilirubin autoxidation have not been identified and characterized, b u t the loss of absorption in t h e visible region during the reaction suggests that extensive degradation of the tetrapyrrole occurs. The development of a strongly positive propentdyopent test during the reaction2 3,2 indicates that the highly water-soluble isomeric water-propentdyopent adducts (5)-(8) (FIGURE 5, R = H) are major products. Therefore, when bilirubin 1X-a is dissolved in water at moderately basic pHs in the presence of air (oxygen) t w o reactions occur, isomerization and autoxidation. The former predominates in more concentrated solutions, the latter under dilute conditions. These reactions should be taken into account during physicochemical studies on bilirubin, for example protein-binding studies, or eliminated b y working under anaerobic conditions. Both of the reactions are so that they are likely t o be inhibited if the bilirubin is bound t o albumin,' of little physiological significance under normal conditions. However, under hyperbilirubinemic conditions, as in neonatal jaundice, where a proportion of the pigment may exist in the free unbound ~ t a t e , ~ 'it, ~is ~most likely that autoxidation of bilirubin will occur. Some of t h e bilirubin derivatives7 that are excreted by jaundiced infants and animals in the absence of a functioning conjugating system are probably autoxidation products, and autoxidation may constitute one of the so-called alternate pathways of bilirubin metabolism. 6924
McDonagh : Bilirubin 1X-a
WAVELENGTH
559
(nml
FIGURE 4. Autoxidation of bilirubin IX-D in 0.05 M-tris buffer, pH 8.5. Initial bilirubin concentration, 15 pM. The solution was prepared under argon, kept for 1 hr in the dark, then bubbled with oxygen for 10 minutes and kept in an oxygen atmosphere in the dark.
M
V M
P
P
(51 M
V
M
P
171 FIGURE 5.
M M
V
Annals New York Academy of Sciences
560
PHOTOCHEMICAL REACTIONS
Pho toisom erization When deoxygenated aqueous solutions of bilirubin 1X-a are irradiated with visible light, the pigment is slowly converted t o a mixture of the 111-, IX- and XIII-a isomers (TABLE 3). In addition t h e bilirubin gradually decomposes t o uncharacterized materials. This photoisomerization reaction is the photochemical analogue of the “dark” radical isomerization already discussed, and both reactions presumably involve the same intermediates and propagation steps. In the photochemical reaction, initiating radicals could be formed by Type I photochemical p r o c e s ~ e sin~ which ~ ~ ~ ~excited triplet state bilirubin 1 x 4 abstracts a hydrogen atom from a second bilirubin molecule. TABLE 3 PHOTOISOMERIZATION OF BILIRUBIN IX-o!* Isomer Composition of Product Medium 0.05 M-tris, pH 8.5 (0.05 M-tris, pH 8.5 0.2 M-phosphate buffer, pH 8.0 chloroform 0.1 M-NaOH 64.2 rM-bovine serum albumin in 0.05 M-tris pH 8.5 Dimethyl sulfoxide
(%I
Bilirubin Recovery (%)
111-0
1X-a
61 79 59 59 64
17 2 21 7 2
61 95 54 83 96
24 10 2
36 35
3 5
94 94
3 2
*
x111-ol 22
31
-
Solutions of bilirubin IX-o! (2.5 mg; 4.3 rmol) in the tabulated media (100 ml) were irradiated under argon for 6 hr through plate glass with a 400 watt high-pressure mercury lamp. Bilirubin was recovered and analyzed by tlc-spectrophotometry. t Control, kept in the dark.
Like the dark reaction, the photochemical isomerization is very sensitive t o the medium. Almost complete isomerization occurs in water at p H 8.0 o r 8.5. In chloroform there is a small degree of photoisomerization. But irradiation of solutions of bilirubin 1X-a in 0.1 N NaOH, aqueous bovine albumin (pH 8.5), or dimethyl sulfoxide does not lead t o significant isomer formation. Prolonged irradiation of depilated, jaundiced (Gunn) rats failed t o produce detectable amounts of the 111- and XIII-a isomers in their serum. The reaction is unlikely t o be physiologically significant (this statement is not meant t o imply that other Type I radical reactions of triplet state bilirubin 1 x 4 are unlikely in vivo).
Pho toaddition Irradiation of bilirubin 1X-a in the presence of alcohols o r thiols under anaerobic conditions results in slow Markovnikoff addition of the alcohol or thiol to the exo-vinyl group of the bilirubin molecule (FIGURE 6 ) . 3 1 - 3 3 An
McDonagh : Bilirubin IX-a
Bilirubin
56 1
Ig. -o(
x = 0,s FIGURE 6. Photoaddition of alcohols and thiols to bilirubin I X a . ionic mechanism has been proposed for this reaction. Compounds which have been shown to add in this way include primary and secondary alcohols,31 2-mercaptoethan01,~~ g l ~ t a t h i o n e , ~and N-acetyl-L-cysteine. Manitto has also reported that when methanol is added to a previously irradiated solution of bilirubin in chloroform, addition of methanol t o the vinyl group still This observation is intriguing because it suggests that bilirubin IX-a may be photochemically converted to a relatively long-lived photoisomer or excited state. However, it is also possible that addition in this case may have been catalyzed by traces of acid9 produced photochemically during the initial irradiation period. Garbagnati and Manitto have suggested that conversion of bilirubin IX-a to more water-soluble and readily excretable tetrapyrroles by photoaddition of nucleophiles may be an important process in phototherapy. 34 A priori, however, it seems unlikely that photoaddition could make an important contribution to the efficacy of phototherapy because the reaction in general appears t o be rather slow and in the presence of oxygen is likely to be overwhelmed by the more rapid photooxidation of the pigment. Photooxidation
The photooxidation of bilirubin IX-a has been extensively studied because of its probable relevance to the mechanism of phototherapy. Although it has been known for a long time that the pigment is readily photooxidized, the products and mechanism of the reaction have been elucidated only recently. Photooxidation of bilirubin 1x4 in ammoniacal methanol yields biliverdin I X a ( 9 ) , methyl vinyl maleimide ( l o ) , hematinic acid imide (1 1) (FIGURE 7), and the isomeric pair of methanol-propentdyopent adducts ( 6 ) and (7) (FIGURE 5); R = CH3) as major p r o d ~ c t s . ~Traces ~ - ~ of ~ the dipyrrylmethme dialdehyde (1 2) are also formed, and a third methanol-propentdyopent isomer has recently been isolated from the photo product^.^" With water as solvent, similar products are obtained except that water-propentdyopent adducts [ ( 6 ) and (7); R = HI are formed instead of the corresponding methanol ad duct^.^^^^* The same compounds are also produced, but at a much greater rate, by sensitized photooxidation of bilirubin 1X-a in methanol or water, using rose bengal as ~ e n s i t i z e r It . ~is~ noteworthy ~~~ that all of the photoproducts that have been definitively identified, especially the propentdyopent adducts, are water-soluble and ought to be rapidly excretable if formed in vivo during phototherapy.
Annals New York Academy of Sciences
562
do
0
H
H
o&o
H
P
P P
OHC
HkL0 H
H
P=CH2CH2COon
FIGURE 7.
The products that are formed on photooxidation of bilirubin IX-a in nonhydroxylic solvents have not been well characterized. In chloroform biliverdin formation may be substantial and greater than in hydroxylic solvent^.^ 8 , 3 9 However, the yields of biliverdin are markedly concentrationdependent, and decrease with decreasing initial concentration of bilirubin I X ~ Y Gray, . ~ ~ Kulczycka, and Nicholson have shown that prolonged exposure of solutions of bilirubin in chloroform to daylight gives several products, including biliverdin, propentdyopents, maleimides, and carboxylic acids.42 Several novel tetrapyrrole pigments (rubins, rhodins, and purpurins) have been isolated in unspecified yield following photooxidation of bilirubin IX-a in pure destabilized chl~roform.~ There is now convincing evidence that bilirubin IX-a photooxidation is mainly a Type I1 p r o c e s ~involving ~ ~ ~ ~singlet ~ oxygen with the overall mechanism shown in FIGURE 8.4 Bilirubin sensitizes the formation of singlet oxygen and then reacts rapidly with it t o give products. Except perhaps for biliverdin IX-a, all of the identified photoproducts are compatible with this m e ~ h a n i s m . They ~ ~ ,apparently ~ ~ ~ ~ ~result from 1,2-addition of singlet oxygen at the enamine-like a and c bridges of the bilirubin IX-a molecule, or 1,4-addition to either of the central pair of pyrrole rings. Rearrangement or solvolysis of the resulting dioxetane or endoperoxide intermediates then yields the observed products. Biliverdin IX-a is not formed by a similar mechanism, but is the product of a separate reaction, which is probably a free-radical, Type I p r o c e s ~ . ~ It ~ .is ~interesting ' ~ ~ ~ t o note that biliverdin IX-tr is a singlet oxygen quencher and, compared t o bilirubin IX-a, is rather stable to p h o t o o x i d a t i ~ n . ~ ~ There are, therefore, a t least t w o pathways for the photooxidation of bilirubin IX-a.One, a Type I1 process involving singlet oxygen, leads directly t o mono- and dipyrroles without involving biliverdin IX-a as an intermediate. The other, probably a Type I reaction, leads initially t o biliverdin IX-a and then
*
344
McDonagh : Bilirubin IX-a
hv
Bi 1 i rubin 1 3
(Bi irubin)* *
b
1
563
* (Bilirubin)
*
3
P (Bilirubin)
3
(Bi irubin) + O2
1 Bi irubin + 0
2
-
______)
1
Bilirubin + O2 Products
FIGURE 8. Singlet oxygen mechanism for bilirubin IX-(u photooxidation.
slowly t o further degradation products. The relative importance of these t w o pathways will vary according to the experimental conditions, particularly solvent, oxygen tension and bilirubin IX-a concentration. This may account for the apparently ’conflicting reports regarding the amount of biliverdin produced on photooxidation of bilirubin (for example, see Reference 46). However, in general, in the presence of air the Type I1 pathway appears t o predominate. Of the two photooxidation pathways, the radical process leading to biliverdin is likely t o be of little significance in phototherapy since biliverdin can be readily ’ radical converted back again to bilirubin enzymatically in v ~ v o . ~Nevertheless, reactions leading to products other than biliverdin could operate during phototherapy. In this regard it should be noted that binding of bilirubin IX-a t o albumin is likely to increase the probability of a Type I mechanism.48 Photooxidation of bilirubin IX-a by the singlet oxygen pathway in vivo seems highly likely, particularly in the skin and peripheral fatty tissues. However, this has not yet been directly demonstrated. Since bilirubin 1x4 is a photosensitizer, one potential risk of phototherapy is photodynamic damage. However, compared to porphyrins and other well known singlet oxygen sensitizers, bilirubin IX-a is a poor photosensitizer. In addition, it reacts very rapidly with singlet oxygen (*lop9 M-’ sec-1);49,50 more rapidly in fact than any other compound yet studied. This combination of properties may well protect the jaundiced child from significant singlet oxygen-induced photodynamic injury during treatment. Formation of singlet oxygen is not likely to be excessive, and any that is formed will tend t o be rapidly scavenged by the bilirubin, which will itself be destroyed in the process.
PHOTOMETABOLISM OF BILIRUBIN I X - ~ l Although much is known about the photochemistry of bilirubin IX-a in vifro, relatively little is known concerning the photochemistry of the pigment in the intact animal. Irradiation of rats or infants with unconjugated hyperbilirubinemia causes a fall in the concentration of the pigment in the serum, but the chemistry underlying this phenomenon is not well understood. Now that phototherapy is so widely used for treating neonatal jaundice, discovering how it works has become a matter of some urgency. Until the mechanism is known it
Annals New York Academy of Sciences
564
will be difficult t o fully assess the safety of the treatment o r lay down effective guidelines for its use. Much of what is known about the mechanism of phototherapy results from studies o n Gunn rats. These are a mutant strain of Wistar rats that have a genetic Homozygous Gunn rats lack a functiondefect in bilirubin ing bilirubin glucuronyl transferase enzyme system and are unable t o conjugate and excrete the pigment in the normal way. Consequently, they have a lifelong unconjugated hyperbilirubinemia and are considered t o be a good model for the human neonate with transient unconjugated hyperbilirubinemia o r permanent unconjugated hyperbilirubinemia (Crigler-Najjar syndrome). Experiments on Gunn ratss3 and jaundiced infantss4 labeled with I4C-bilirubin have shown that there is an enhancement in the excretion of radioactive label when the subject is exposed to visible light. Of the total amount of label excreted, the greatest proportion is into the bile, and, as first shown by O ~ t r o w , ~the effect of, phototherapy on biliary excretion is particularly striking. FIGURE 9 shows the changes in the excretion of radioactivity by a labeled Gunn rat during a light-dark-light cycle. The method was essentially the same as O s t r o w ’ ~ .Prior ~ ~ t o the experiment the rat was shaved dorsally and injected with a pulse of biosynthetic l4C-bilirubin IX-a26 and then left in the dark for about twelve hours to allow exogenous labeled pigment to equilibrate with endogenous material. Bile was collected continuously throughout the experiment via a polythene tube inserted into the bile duct and fractions were taken every four hours for counting. During the “light” period the creature, with head covered, was irradiated with visible light from a fluorescent light fixture (Duro-Test Vitalite). Even in the dark there is some excretion of bilirubin-derived radioactivity. This is not due t o excretion of bilirubin IX-a itself, but to unidentified
4
I
LIGHT
j
I
E:
? P
u
10
HOURS AFTER
“C
INJECTION
FIGURE 9. Total biiiary excretion of 14C by a male hornozygous Gunn rat during a dark-light-dark cycle. Light was from Vitalite fluorescent tubes delivering an illumination of 500 foot-candles at the rat’s back.
McDonagh : Bilirubin IXiv
565
derivativest which, as discussed earlier, may include water-soluble autoxidation products. immediately following irradiation there is a very pronounced increase in isotope excretion, which gradually abates after the light is extinguished. Significantly, throughout the entire experiment the bile flow remained essentially constant, indicating that the photobiological response reflects an increased output of isotope and not merely an increase in the biliary concentration of the isotope. As originally shown by O s t r o ~ , ~ the light-induced increment in the amount of radioactive material excreted is made up of t w o components. One component contains unidentified bilirubin derivatives and the other consists of unconjugated bulirubin IX-a. The bilirubin component is substantial5 and may account for u p t o 60% of the photostimulated excretion of radioactivity. Recent experiments suggest that phototherapy causes qualitatively similar effects in jaundiced ~ h i l d r e n . ~ Phototherapy, therefore, is effective in reducing serum bilirubin levels because it causes biliary excretion of the pigment itself and compounds derived from it. At present, it is a mystery how light stimulates the excretion of unconjugated bilirubin; possibly this is not due t o a direct photochemical reaction at all. Since none of the other derivatives that accompany the bilirubin have been identified, their origin, too, remains obscure. However, it is likely that these compounds are derived, a t least in part, from photooxidation of bilirubin in t h e skin and extravascular tissue. As previously pointed out, bilirubin IX-a is a rather poor photosensitizer. In vitro, its photooxidation is accelerated by the addition of better singlet oxygen sensitizers such as rose bengal, methylene blue, or p ~ r h y r i n s . ~ These observations suggested that by using photosensitizers in vivo it might be possible to magnify the effect of light in reducing serum bilirubin concentrations. FIGURE 10 shows the effect of intravenously administered “hematoporphyrin” ,4495
Serum
Bilirubin (%A] 30
It1
20 10 0 10 20
30
(-1
40
----- wiinoui
50
-
60
HEHATOPORPHYRIN
AFTER HEMATOPORPHYRIN
70
80
TIME (hrr)
FIGURE 10. Effect of hematoporphyrin on the efficacy of phototherapy in a Gunn rat. The rat was injected (i.v.) with saline solution and subsequently hematoporphyrin (0.5 mg/100 g) at t = 0. After 1 hr the rat was irradiated (Vitalite) for 5.5 hr and then kept in the dark for > 65 hr. Serum bilirubin values were determined by the diazo method on tail-vein blood samples and are expressed as a percentage of change from the serum bilirubin concentration at the start of the experiment.
t See Reference 43 for evidence regarding the structure and origin of some of these derivatives.
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Annals New York Academy of Sciences
on the efficacy of phototherapy in a Gunn rat.$ Brief irradiation of the animal (dorsally shaved) with a Vitalite fixture produced only a modest decrease in the serum bilirubin concentration, whereas a similar period of irradiation following injection of the animal with “hematoporphyrin” resulted in a pronounced drop in the serum bilirubin level (FIGURE 10). Hematoporphyrin had no effect on serum bilirubin levels when the animal was kept in the dark. A similar effect has been noted b y Kostenbauder and Sanvordeker using exogenously administered riboflavin as photosensitizer. These experiments d o not prove that a singlet oxygen Type I 1 mechanism is involved in phototherapy, although they lend credence t o the possibility. However, they d o suggest that endogenous photosensitizers such as porphyrins or riboflavin might play some part in causing the dissappearance of bilirubin 1X-cy during phototherapy. SUMMARY
In solution in the dark bilirubin IX-(r readily undergoes autoxidation and isomerization. The latter can occur by an ionic or a free radical process, depending on the conditions. Irradiation of bilirubin IX-a in vitro can lead to photoisomerization, photoaddition of alcohols and thiols, o r photooxidation. Photooxidation can proceed by two pathways: one is a selfsensitized singlet oxygen process leading rapidly to mono- and dipyrroles, and the other leads initially t o biliverdin and may involve a free radical mechanism. lrradiation of jaundiced rats causes biliary excretion of the pigment and enhances the excretion of bilirubin derivatives. The overall effect is t o lower the concentration of bilirubin in the serum. This effect can be enhanced by administration of appropriate photosensitizers.
ACKNOWLEDGMENTS
I wish t o thank Mr. Francis K. Assisi and Mrs. Lucita A. Palma for their technical assistance, and Professor R. Schmid for his cooperation and support. This work was aided by generous support and equipment given b y the Duro-Test Corporation, North Bergen, New Jersey. REFERENCES 1. SCHMID, R. 1972. Hyperbilirubinemia. In The Metabolic Basis of Inherited Disease, 3rd Ed. J. B. Stanbury, J. B. Wyngaarden & D. S. Frederickson, Eds. 1141-1178. McGraw-Hill, Inc. New York, N.Y. 2. THALER, M. M. 1972. Neonatal hyperbilirubinemia. Seminars Hematol. 9 ( 2 ) : 107-112. 3. DIAMOND, I. 1969. Bilirubin binding and kernicterus. Advan. Pediat. 16: 99-1 19. 4. LUCEY. J. F. 1972. Neonatal phototherapy: uses, problems and questions. Seminars Hematol. 9 (2): 127-135. $ The hematoporphyrin used was commercial material treated with acetic acid-sulfuric acid according to the method of Lipson and Balde~.~’Subsequent work has shown this material to be a mixture of porphyrins containing hematoporphyrin as a major constituent.
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5. SCHMID, R. 1971. More light on neonatal hyperbilirubinemia. New Eng. J. Med. 285: 520-522. 6. Editorial. 1972. Phototherapy in neonatal jaudice. Brit. Med. J. 2: 62-63. 7. SCHMID, R. & L. HAMMAKER. 1963. Metabolism and disposition of C14-bilirubin in congenital nonhemolytic jaundice. J. Clin. Invest. 42: 1720-1734. 8. MCDONAGH, A. F. & F. ASSISI. 1972. Direct evidence for the acid-catalyzed isomeric scrambling of bilirubin IX-a. J. Chem. SOC.Chem. Commun.: 117-119. 9. MANITTO, P. & D. MONTI. 1973. Acid-catalyzed addition of alcohols and thiols t o bilirubin. Experientia 29: 137-139. 10. MCDONAGH, A. F. & F. ASSISI. 1971. Commercial bilirubin: A trinity of isomers. FEBS Lett. 18: 315-317. 11. MCDONAGH, A. F. & F. ASSISI. Unpublished observations. 12. MAUZERALL, D. 1960. The thermodynamic stability of porphyrinogens. J. Amer. Chem. SOC.82: 2601 -2605. 13. JACKSON, A. H., G. W. KENNER & J. WASS. 1972. Pyrroles and related compounds. Part XX. Synthesis of Coproporphyrins. J. Chem. SOC. Perkin Trans. 1.: 1475-1483. 14. BONNETT, R. & A. F. MCDONAGH. 1970. The isomeric heterogeneity of biliverdin dimethyl ester derived from bilirubin. J. Chem. SOC.,D: 238-239. 15. BONNETT, R. & A. F. MCDONAGH. Unpublished observations. 16. MCDONAGH, A. F. & F. ASSISI. 1972. The ready isomerization of bilirubin IX-or in aqueous solution. Biochem. J. 129: 797-800. 17. CHEUNG, W. H., A. SAWITSKY & H. D. ISENBERG. 1966. The effect of bilirubin on the mammalian erythrocyte. Transfusion 6: 475-486. 18. ZETTERSTROM, R. & L. ERNSTER. 1956. Bilirubin, an uncoupler of oxidative phosphorylation in isolated mitochondria. Nature 178: 1335-1337. 19. VOGT, M. T. & R. E. BASFORD. 1968. The effect of bilirubin on the energy metabolism of brain mitochondria. J. Neurochem. 15: 1313-1320. 20. MUSTAFA, M. G., M. L. COWGER & T. E. KING. 1969. Effects of bilirubin on mitochondria1 reactions. J. Biol. Chem. 244: 6403-6414. 21. TAPPEL, A. L. 1973. Lipid peroxidation damage t o cell components. Fed. Proc. 32: 1870-1874. 22. COLEMAN, P. S. 1973. Uncoupling of oxidative phosphorylation by a stable free radical and its diamagnetic homolog. Riochim. Biophys. Acta 305: 179-184. 23. BINGOLD, K. 1935. Weitere untersuchungen zur formulierung eines biologischchemischen blutkreislaufes. Klin. Wochenschr. 14: 1287- 1289. 24. FOG, J. & B. BUGGE-ASPERHEIM. 1964. Stability of bilirubin. Nature 203: 756-757. 25. MCDONAGH, A. F. & L. PALMA. Unpublished observations. 26. OSTROW, J. D., L. HAMMAKER & R. SCHMID. 1961. The preparation of crystalline bilirubin-C14. J. Clin. Invest. 40: 1442-1452. 27. ODELL, G . B. 1959. The dissociation of bilirubin from albumin and its clinical implications. J. Pediat. 55: 268--279. 28. NAKAMURA, H. & R. LARDINOIS. 1972. Unbound bilirubin in icteric newborns. Biol. Neonate 21: 400-417. 29. SCHENCK, G . 0. 1963. Photosensitization. Ind. Eng. Chem. 55: 40-43. 30. GOLLNICK, K. 1968. Type 11 photooxygenation reactions in solution. Advan. Photochem. 6: 1-22. 31. MANITTO, P. 1971. Photochemistry of bilirubin. Experientia 27: 1147-1 149. 32. MANITTO, P. & D. MONTI. 1972. Photoaddition of sulphydryl groups t o bilirubin in vitro. Experientia 28: 379-380. 33. MANITTO, P., D. MONTI & E. GARBAGNATI. 1972. Photochemical addition of N-acetyl-L-cysteine and glutathione to bilirubin in vitro and its relevance to phototherapy of jaundice. Farmaco, Ed. Sci. 27: 999-1002. 34. GARBAGNATI, E. & P. MANITTO. 1973. A new class of bilirubin photoderivatives obtained in vitro and their possible formation in jaundiced infants. J. Pediat. 83: 109-1 15.
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35. LIGHTNER, D. A. & G. B. QUISTAD. 1972. Methylvinylmaleimide from bilirubin photooxidation. Science 175: 324. 36, LIGHTNER, D. A. & G. B. QUISTAD. 1972. Hematinic acid and propentdyopents from bilirubin photooxidation in vitro. FEBS Lett. 25: 94-96. 37. LIGHTNER, D. A. & G. B. QUISTAD. 1972. h i d e products from photo-oxidation of bilirubin and mesobilirubin. Nature New Biol. 236: 203 -205. 38. BONNETT, R. & J. C. M. STEWART. 1972. Photo-oxidation of bilirubin in hydroxylic solvents: propentdyopent adducts as major products. J. Chem. SOC. Chem. Commun.: 596. 39. LIGHTNER, D. A., D. C. CRANDALL, S. GERTLER & G. B. QUISTAD. 1973. On the formation of biliverdin during photooxygenation of bilirubin in vitro. FEBS Lett. 30: 309-312. 40. BONNETT, R. & J. C. M. STEWART. Personal communication. 41. BONNETT, R. & J. C. M. STEWART. 1972. Singlet oxygen in the photo-oxidation of bilirubin in hydroxylic solvents. Biochem. J. 130: 895-897. 42. GRAY, C. H., A. KULCZYCKA & D. C. NICHOLSON. 1972. The photodecomposition of bilirubin and other bile pigments. J. Chem. Soc. Perkin Trans. I.: 28 8 -294. 43. BERRY, C. S., J. E. ZAREMBO & J. D. OSTROW. 1972. Evidence for conversion of bilirubin to dihydroxyl derivatives in the Gunn rat. Biochem. Biophys. Res. Commun. 49: 1366-1375. 44. MCDONAGH, A. F. 1971. The role of singlet oxygen in bilirubin photo-oxidation. Biochem. Bioph.ys. Res. Commun. 44: 1306-1311. 45. MCDONAGH, A. F. 1972. Evidence for singlet oxygen quenching by biliverdin I X a dimethyl ester and its relevance to bilirubin photo-oxidation. Biochem. Biophys. Res. Commun. 48: 408-415. 46. DAVIES, R. E. & S. J. KEOHANE. 1970. Some aspects of the photochemistry of bilirubin. Boll. Chim. Farm. 109: 589-598. 47. TENHUNEN, R., M. E. ROSS, H. S. MARVER & R. SCHMID. 1970. Reduced nicotinamide-adenine dinucbotide phosphate dependent biliverdin reductase: partial purification and characterization. Biochemistry 9: 298-303. 48. BELLIN, J. S. 1968. Photophysical and photochemical effects of dye binding. Photochem. Photobiol. 8: 383-392. 49. LEE, J., 1. B. C. MATHESON, J. E. WAMPLER, R. D. ETHERIDGE & N. U. CURRY. 1973. Inhibition of laser generated singlet oxygen reaction with bilirubin by superoxide dismutase. Fed. Proc. 32: 661 (abstract). 50. FOOTE, C. S. Personal communication. 51. GUNN, C. H. 1938. Hereditary acholuric jaundice in a new mutant strain of rats. J. Heredity 29: 137-139. 52. SCHMID, R., J. AXELROD, L. HAMMAKER & R. L. SWARM. 1958. Congenital jaundice in rats due to a defect in glucuronide formation. J. Clin. Invest. 37: 1123-1130. 53. OSTROW, J. D. 1971. Photocatabolism of labeled bilirubin in the congenitally jaundiced (Gunn) rat. J. Clin. Invest. 50: 707-718. 54. CALLAHAN, E. W., M. M. THALER, M. KARON, K. BAUER & R. SCHMID. 1970. Phototherapy of severe unconjugated hyperbilirubinemia: formation and removal of labeled bilirubin derivatives. Pediatrics 46: 841 -848. 55. LUND, H. T. & J. JACOBSEN. 1972. Influence of phototherapy on unconjugated bilirubin in duodenal bile of newborn infants with hyperbilirubinemia. Acta Paediat. Scand. 61: 693-696. 56. CREMER, R. J., P. W. PERRYMAN & D. H. RICHARDS. 1958. Influence of light on the hyperbilirubinaemia of infants. Lancet 1: 1094- 1097. 57. LIPSON, R. L. & E. J. BALDES. 1960. The photodynamic properties of a particular hematoporphyrin derivative. Arch. Dermatol. 82: 508-5 16. 58. KOSTENBAUDER, H. B. & D. R. SANVORDEKER. 1972. Riboflavin enhancement of bilirubin catabolism in vivo. Experientia 29: 282-283.
