Eur. J. Biochem. 72, 213-221 (1977)

Photooxidation of Human Serum Albumin and Its Complex with Bilirubin Anders Overgaard PEDERSEN, Fritz S C H q N H E Y D E R , and Rolf BRODERSEN Institute of Medical Biochemistry, University of Aarhus (Received May 20iOctober 14, 1976)

Irradiation with visible light of human serum albumin in aqueous solution at p H 8, in the presence of catalytic amounts of rose bengal or methylene blue, resulted in random oxidation of the histidine residues in the protein under consumption of one mole 0 2 , and release of somewhat less than one proton, per histidine residue degraded. An increase of light absorption at 250 nm was proportional to the amount of oxygen consumed. Bilirubin bound to the oxidized protein showed an increased light absorption at its maximum, 460 nm, and a decreased binding affinity, indicating a conformational change of the protein on oxidation of histidine residues. This change also resulted in a slight perturbation of tyrosine light absorption, corresponding to a shift of the chromophore to more polar surroundings. Further, a sensitized oligomerization of albumin was observed, independent of oxidation of the histidine residues, and not consuming oxygen. Irradiation of a complex of human serum albumin with one molecule of bound bilirubin, in the absence of a sensitizing dye, resulted in a fast, non-oxygen consuming process whereby the light absorption maximum of the pigment was shifted 4 nm towards longer wavelength and part of the bilirubin was converted to a more polar pigment, bound less firmly to the protein. This was followed by a relatively slow oxidation of the pigment under uptake of one mole 0 2 . Parallel photooxidation of the protein carrier could not be detected. It is considered possible that the fast, anaerobic process is operative in phototherapy of hyperbilirubinemia in the newborn. Serum albumin is probably not oxidized during this treatment. Photooxidation of proteins in the presence of sensitizers, such as rose bengal or methylene blue, generally involves a singlet-oxygen attack on histidine residues [l 1. Also the naturally occurring pigment, bilirubin, is able to transfer excitation energy to molecular oxygen [2,3]. Extensive use of phototherapy in newborn infants with hyperbilirubinemia has stimulated research in this field and the question has been raised whether bilirubin is photooxidized in the skin [4] during phototherapy, and whether it also sensitizes photooxidation of serum albumin to which bilirubin is bound in the blood plasma [5,6]. This paper deals with the stoichiometry and kinetics of photooxidation of human serum albumin, sensitized by rose bengal or methylene blue, and with changes caused by irradiation of the bilirubin . albumin complex. Mechanisms in phototherapy of hyperbilirubinemia are discussed. The experimental material in this paper was taken from the Ph. D. thesis of A.O.P. Enzyme. Peroxidase or donor: H202 oxidoreductase (EC 1.11.1.7).

MATERIALS AND METHODS

Chemicals Human serum albumin was from AB Kabi, Sweden. The preparation contained 98% serum albumin, and 0.5- 1.0 mol fatty acids per mol albumin Desalting on a mixed-bed ion-exchange column, defatting with charcoal in acid solution [7], and isolation of the protein monomer was done in a few experiments. We found photooxidation to be independent of these procedures, and the commercial preparation was then used as such. Two sensitizers were used. Rose bengal (BDH, cat. 1 cm, 534 nm) = 403 in phosno. 20103) had A (1 phate buffer, pH 8.0. This molecule carries one negative charge at pH above 4 and is firmly bound to albumin [S]. Methylene blue (Merck, cat. no. 1283) had A (1 %, 1 cm, 666 nm) = 1780 in phosphate buffer, pH 8.0. This cationic molecule is not bound to albumin. Bilirubin (Sigma, cat. no. B 4126) had A (1 %, 1 cm, 460 nm) = 1030 in chloroform.

x,

Photooxidation of Albumin and Bilirubin

214

Pho tooxidation

Albumin solutions for photooxidation contained the protein, 30 pM, and sensitizer, 3 pM, in 50 mM phosphate buffer, pH 8.0. The process was conducted in closed glass flasks under eight fluorescent lamps, Osram 20 Wjl5, with emission between 400 and 700 nm. Irradiation of bilirubin . albumin was carried out with 0.9 mol bilirubin per mol albumin, the latter in the concentration 15 pM, in a phosphate buffer, 0.1 M, pH 7.5. The oxygen concentration in the reaction mixture markedly influenced the rate of oxidation when pO2 was below 150 Torr (20 kPa). To ensure saturation kinetics with respect to oxygen throughout the period of irradiation, oxygen gas was introduced in sufficient amounts to maintain p 0 2 higher than this level throughout the period of irradiation. Fractionation of the Reaction Mixture

Photooxidized protein was isolated from reaction mixtures, containing methylene blue, by gel filtration or by treatment with charcoal at pH 3. Isolation of albumin after irradiation of its bilirubin complex was done by treatment of the reaction mixture with charcoal in the presence of salicylic acid. Bilirubin remaining after irradiation of the bilirubin . albumin complex, could be recovered by addition of a buffer containing salicylate and ascorbate, followed by chloroform extraction at p H 8 ~91. Analytical Procedures. Characterization of Products

Oxygen tension was measured by a thermostated electrode (Radiometer E-5046). Consumption of oxygen in the closed one-phase system was calculated from these data. Spectra were recorded on Unicam SP-800 or Beckman DB spectrophotometers. The ultraviolet difference spectra were obtained with a Beckman Acta CV spectrophotometer. Proton release during photooxidation was titrated with NaOH in non-buffered medium. Amino acid composition was examined, after acid hydrolysis (6 M HCl, 110 "C, 20 h), using a Beckman model 120 C amino acid analyzer. Tryptophan was determined according to Spies & Chambers [lo] after partial digestion with chymotrypsin and trypsin, as suggested by Harrison & Hofman [ll]. A series of histidine determinations were done after irradiation of bilirubin . albumin, according to Sokolovsky & Vallee [ 121. Gel filtration was done on Sephadex G-150, column length 90 cm, eluant 1 "/, NaCI. Ion exchange

was done on DEAE-Sephadex A50, column length 87 cm, elution with 0.13 M piperazine, pH 5.0. Binding affinity of bilirubin to the isolated protein was studied by measuring the rate of oxidation of bilirubin with hydrogen peroxide, catalyzed by horse radish peroxidase. The initial velocity is proportional to the equilibrium concentration of free bilirubin [13]. RESULTS DYE-SENSITIZED PHOTOOXIDATION OF ALBUMIN

Spectral Changes and Oxygen Consumption

Absorption spectra of human serum albumin, on irradiation with visible light in the presence of methylene blue or rose bengal, showed gradual increases of light absorption at wavelengths below 280 nm and around 325 nm (Fig.1A). These changes were irreversible and independent of which sensitizer was used. After discontinuation of irradiation the spectra remained constant. The increase of light absorption at 250 nm, was proportional to the oxygen consumption, as seen from Fig. 1 B. The linearity of light absorption at 250 nm and oxygen uptake formed the basis for convenient spectrophotometric monitoring of the photooxidation. It was found that the rate was independent of the oxygen concentration at the levels used. During the early part of the process, until 6-8 mol 0 2 was consumed per mol albumin, the rate was proportional to 15.4 less the consumed amount of oxygen. Since the albumin molecule contains 16 histidine residues, this course corresponded to random oxidation of all, or nearly all histidines. Influence o f p H

The increase of light absorption at 250 nm varied with the pH of the medium, as seen from Fig. 2 (1 h of irradiation, about 8 rnol 0 2 consumed at pH 8). The function was sigmoidal with an inflexion point at pH 7, consistent with photooxidation of deprotonized histidine groups. The zero rate at low pH values indicated protection .of all 16 histidine residues, in agreement with complete protonization of these residues at pH 4. Amino Acid Analysis. Release of Protons

The amino acid content of photooxidized albumin specimens is seen in Table 1 . Histidine content declined during the process of photooxidation. Among other amino acids, tyrosine and tryptophan decreased slightly. As seen from Fig. 3 there was stoichiometric degradation of one mole histidine for each mole oxygen, until five. In buffer-free medium a decrease of pH was observed. Titration to constant pH of 8.0 showed a progressive release of protons (Fig. 3).

A. 0. Pedersen, F. Sch$nheyder, and R. Brodersen

215

Lrradiat ion t irne ( rnin) 120 ~

Wavelength (nrn)

Fig. 1. ( A ) Ultraviolet absorption spectru o/ tilhumin afier wrying periods ofisrudiu[iot1in the presence of rose hengal. (B)Oxygen uptake related 10 he increase of absorption at 250 nm. With rose bengal as a sensitizer, the slope of the line in (B) was (2.82 5 0.03) x 10’ (mol albumin x rnol O2 x cm)-’. The coefficient of correlation was ? = 0.998. With methylene blue, r2 was 0.989 and the slope was not significantly different from that obtained with rose bengal. Sensitizer: (0)methylene blue, (0)rose bengal.

0

0

0

f

0

0

2 0.5

e.

0

~

0

’

:::,.,.,> . 5

10

~

Fig. 2. Senstti:etl pliofoo.\-i(u,ionu/io~i o j PH ulbunirii as a f’unctiun Of’ p H .

“-1

-0

a

5

After 1 h of irradiation at the pH value indicated, the solutions were brought to pH 7 and absorption was measured. Sensitizers as in Fig. 1

I

5

0

It is concluded that during the first part of sensitized photooxidation of human serum albumin at pH 8, until about 6 - 8 mol oxygen have been consumed per mol protein, the dominating process is oxidation of histidine residues. 1 mol 0 2 is consumed for each histidine degraded, and somewhat less than 1 mol H + is released. All 16, or nearly all, histidine residues in the albumin molecule are oxidized with equal velocity. All histidines can be protonated and therefore protected against oxidation by lowering the pH to about 4- 5.

I )

10

0 2 uptake ( m o l / m o l albumin)

Fig. 3. Histidirir ( . o t i f ~ i iii i urid relc~usc~ of prorori.c 10, 0)during photooxidation of’alhumin. The oxygen uptake was calculated from the increase of light absorption at 250 nm. The dotted line is drawn with slope = - 1 and intercepl = 16. Sensitizers as in Fig. 1

Chromatographic Analysis

Gel chromatography of non-oxidized serum albumin showed the presence of a small amount of oligomer protein, besides the monomer peak. After

Photooxidation of Albumin and Bilirubin

216

Table 1. Amino acid composition of human serum albumin after varying degrees of photooxidation, indicated by oxygen uptake Numbers in paranthesis refer to non-irradiated albumin and are taken from Meloun el al. [14] Amino acid composition with __ _--0

Amino acid

0 2

uptake of - -~

-

mol/mol protein ~

Aspartic acid or asparagine Threonine Serine Glutaniic acid or glutamine Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine P henylalanine Lysine Histidine Arginine Tryptophan

-

---

.

1 7 mol/mol

. .

~

-

~~

~

-

4 8 mol/mol

__

-

~

(53) (28) (24)

53.9 25.8 19.3

54.5 25.7 19.2

54.1 26.6 21.1

54.4 26.4 21.2

54.4 26.9 21.5

54.4 26.9 21.7

(82) (24) (12) (62) (35) (41) (6) (8) (61) (18) (31) (59) (16) (24) (1)

81.9 24.1 15.1 63.9 26.2 40.4 3.4 1.3 60.3 15.5 29.7 60.4 15.9 22.1

82.5 24.1 11.2 64.1 21.0 40.0 3.9 6.9 60.6 14.5 28.4 60.8 15.4 22 2

82.9 23.7 11.7 63.1 29.2 40.2 4.8 7.4 60.6 14.8 29.2 60.0 13.6 23.0

84.0 24.7 10.6 63.3 26.0 39.3 4.0 6.7 61.5 13.1 27.5 61.0 13.1 22.0

83.9 24.5 11.1 63.7 27.8 39.8 4.2 6.5 60.3 13.4 28.0 60.6 10.2 22.4

83.9 24.3 11.4 64.0 29.7 39.7 4.2 6.5 59.8 13.5 28.3 60.6 10.7 22.4

59.5 59.4 59.6 15.9 16.0 16.2 23.5 23.6 23.3 1.0 1.1

-

- -~ ~8 4 molimol

-

__

-.

..

-.

59.6 59.4 59.6 8.6 8.3 8.5 23 4 23.6 23.4 0.8 0.8

I " Irradiation time (min)

- 50

-

8

1oc

t

t

>

Elution volume

250

I

I

300 Wavelength ( n m )

350

)

Fig.4. ( A ) Gel chromatography ofphotooxidized albumin on Sephade.x G-150 and ( B ) light absorption spectra of the polymer i x ) and monomer (0) fractions. (A) Sensitizer, methylene blue; irradiation times as indicated; eluant, 1 NaCI. Hatching indicates the oligomer (left) and monomer (right) fractions collected. (B) Irradiation time, 120 min. The polymer spectrum was amplified to obtain equal absorptions at 280 nni

A. 0. Pedersen, F. Schqhheyder, and R. Brodersen

211 O2 ( m o l / mol albumin)

12.5 \ 11.8

I

I

I

I

350

400

450

500

>

Wavelength (nrn)

Fig. 5. Difference spectra of bilirubin udded

10

photoo.dili-rd rill~uminagaimr bilirubin udded to non-oxidized albumin

this case absorption spectra indicated that all three fractions were oxidized to the same extent.

i:

1 I 0

Structural Changes, Studied with Bilirubin as a Probe

0'

I

5

I >

10

02 (rnolirnol albumin)

Fig. 6 . Velocity of perolida.~e-c.ataly_.edoxidation of bilirubin added 10 phoroox-idixd ulbumin

irradiation in the presence of oxygen and methylene blue, the amount of oligomer was increased (Fig. 4A). Absorption spectra of monomer and oligomer fractions (Fig.4B), were multiplied by a factor to obtain equal ordinates at 280 nm. Differences in the degree of oxidation of the fractions would then result in differences of absorbance below 280 and around 325 nm. Such differences could not be observed and it is concluded that the oligomer and monomer fractions were oxidized to the same degree. Apparently oligomerization proceeds by a mechanism independent of the oxidation of histidine residues, and does not consume oxygen. Chromatography on DEAE-Sephadex was performed at a pH close to the isoelectric point of albumin, with the aim of separating products with different electric charge. With photooxidized albumin three poorly resolved fractions were obtained, but even in

After photooxidation of albumin during varying lengths of time, bilirubin was added, 1 mol per rnol albumin, and absorption spectra of the bilirubin chromophore were studied. The changes observed were complex and are pictured as the difference spectra (Fig. 5). It is seen that the absorption initially increased around 467 nm, where the maximum of bilirubin bound to albumin is located. No shift of wavelength of the maximum was seen early in the course. Later on a blue shift occurred, beginning in the interval between 6.4 and 9.9 mol oxygen consumption, as visualized by the unset of decreasing light absorption at 495 nm while the increase continued at wavelengths below the maximum. In order to gain some insight into the structural background for this dual change of spectrum, the experiments referred to in Fig.5 were repeated in the presence of 5 M urea. In this case we observed a clean blue shift of the bilirubin chromophore with isosbestic point at 455 nm, identical to spectra of two-component mixtures of free and albumin-bound bilirubin. The late blue shift (Fig. 5) may thus be explained by release of bilirubin from the binding site. The early increase of the bilirubin maximum may be due to a conformational change of the albumin molecule. This change did not take place during photooxidation, when 5 M urea was present, and therefore seems to involve structures which are unfolded by 5 M urea. Binding affinity of photooxidized albumin for bilirubin was further investigated by determination of the rate of oxidation of the ligand with hydrogen peroxide, catalyzed by peroxidase. Free bilirubin is readily oxidized under these circumstances while the

218

Photooxidation of Albumin and Bilirubin

pigment is protected when bound to native albumin [13]. As seen in Fig.6, photooxidation of albumin resulted in increased susceptibility of bilirubin, added after photooxidation. The change was perceptible from the beginning of photooxidation and became very pronounced at high degrees of oxidation of the protein. These observations are consistent with a gradual decrease of bilirubin binding affinity during the early change of conformation, followed by release of bilirubin and loss of the binding site later in the course.

Spectral Perturbation of Tyrosine Chromophores The spectral changes during the early part of photooxidation were further investigated around 280 290 nm. Difference spectra, obtained with photooxidized albumin (0 - 6 mol O2 consumed) in the reference and a non-irradiated specimen in the sample beam, showed progressive development of maxima at 281 and 288 nm, corresponding to a blue shift of tyrosine absorption. The early conformational change thus involves an area in the albumin molecule where tyrosine is located.

IRRADIATION OF THE BILIRUBIN . ALBUMIN COMPLEX

Chemical Changes and Oxygen Consumption

A solution of equimolar amounts of human serum albumin and bilirubin in phosphate buffer at pH 7.5, in which most of the pigment is bound to one site on

0 1

the protein [15], was irradiated under aerobic conditions, as used for photooxidation of albumin. No sensitizer was added. Visible spectra were recorded, as seen in Fig. 7. The following changes were observed. a) A rapid red shift by 4 nm took place in less than 30 s, without consumption of oxygen. b) The major change occurred during 3 h, with formation of a maximum at 370 nm and isosbestic point at 388 nm, a marked decrease of absorption in the region of the bilirubin maximum, and a slight increase at wavelengths above 520 nm. This change was parallelled by consumption of one mole 0 2 . c) A decrease of absorption at about 320- 420 nm, whereby the maximum at 370 nm disappeared, was seen late in the course from 3 to about 24 h. Spectral changes from 500- 700 nm were insignificant. The protein was isolated, after photolysis in the presence of bilirubin for 0, 10, and 60 min. These purified albumin preparations could not be distinguished by their ultraviolet absorption spectra, by their histidine and tryptophan content, and by their ability to ligand bilirubin. In addition, the rate of photooxidation (step 2) was insensitive to pH in the region 5.5 - 10,and no release of protons could be detected. Extraction with Non-aqueous Solvents After 1 min of irradiation of bilirubin . albumin, followed by chloroform extraction, the light absorption of the extract was decreased to 0.7 of the nonirradiated reference. The spectrum of the pigment in the chloroform phase was identical to that of bili-

,

I

I

350

400

450

I

500

Wavelength (nrn)

Fig. I. Absorption spectra qf'un equimolur mi.\-turc. of' brliruhin urrd ~ N ~ L I I I I in I I Ipliosphute buffer, p H 7.5, qfier wrying periods (. . . . ' .) Calculated difference spectrum between 0 and 30-s sample

of' irradiution

A. 0. Pedersen, F. SchQnheyder, and R. Brodersen

219

*L 302\/

H

*

S e n s

3Sens

L

H

Scheme 1. Tentative mechanism for sensitized photooxiduiion ojhistidine residues in humiur sorum albumin. Physical reactions: excitation of the sensitizer (singlet ground state) with visible light to its first excited singlet state followed by intersystem-crossingto its first excited triplet state; transfer of the excitation energy to molecular oxygen (triplet ground state) results in ground state sensitizer and molecular oxygen excited to its singlet state. Chemical reactions: cycloaddition of singlet oxygen to the imidazole ring resulting in an unstable cyclic peroxide braking down to a hydroxy-imidazolone intermediate,finally reformation of the aromatic system via proton rearrangement is depicted

rubin. The aqueous remains contained a pigment with light absorption maximum at 440 nm. Rate of Peroxidase Oxidation of the Irradiated Complex

Irradiated solutions of the bilirubin . albumin complex were subjected to peroxidase-catalyzed oxidation with hydrogen peroxide. The initial rate of the peroxidase process was considerably increased, as a result of a few minutes of photolysis, preceding the enzymic oxidation. Similar observations have been reported previously [161. Conclusions The above findings indicate that albumin does not undergo photooxidation in the presence of bilirubin. This is seen from the unchanged ultraviolet spectrum and the constant amount of histidine found. The fact that only one molecule of oxygen was consumed during 3 h of photolysis further excludes bilirubinsensitized photooxidation of albumin, since at least 1 molO2 is consumed by oxidation of bilirubin. It is interesting to note that a photo-induced change in the bilirubin . albumin complex took place during less than 1 min of irradiation. No oxygen was consumed, but 0.3 of the bilirubin was converted to a pigment which differed from bilirubin with respect to absorption spectrum, chloroform solubility and sensitivity to enzymic oxidation in the presence of albumin. DISCUSSION Previous investigators have demonstrated the reactivity of histidine groups in photooxidation of proteins [l], and also in the case of human serum albumin [17]. It has been reported that each histidine reacts with 1 mol oxygen [18]. The present observations confirm this stoichiometry and show that histi-

dine is the only amino acid oxidized until more than 6 mol oxygen have been consumed. Stoichiometric release of protons has previously been observed during photooxidation of pure histidine [191. Somewhat less than 1 mol hydrogen ion was titrated in our experiments on oxidation of each mol histidine in albumin. The course of events is independent of whether an albumin-bound sensitizer (rose bengal) or one freely dissolved in the medium (methylene blue) is used. This seems to exclude a direct reaction between excited sensitizer and protein and is in agreement with a process mediated through a common intermediate, singlet oxygen. The mechanism can tentatively be formulated as a cyclo-addition of singlet oxygen to the imidazole group, as suggested by Tomita et al. [20] and Wasserman [21], followed by rearrangement, and release of a proton as pictured in Scheme 1 . Spectral changes during photooxidation of histidine-containing peptides and proteins have been described [22 - 241. The present observations show that the increase of light absorption at 250 nm is proportional to the oxygen uptake, until about 8 mol 0 2 has been consumed. The kinetics of this part of the oxidation is compatible with equal reactivity of all histidine residues in the albumin molecule. This finding does not exclude protection of one or two histidines but does rule out the existence of groups with specific high reactivity. Partial oxidation of histidine residues at random should result in a heterogeneous mixture of products and it was accordingly not possible to isolate a well-defined protein by ionexchange chromatography. Some insight into the average structural changes on photooxidation was obtained by studying properties of the complex with bilirubin, added after oxidation of the albumin. Sensitized photooxidation of less than six histidines resulted in a conformational change close to the primary binding site for bilirubin, and a decrease of bilirubin binding affinity. A structural change was also seen from a progressive per-

220

turbation with blue shift of one or a few tyrosine groups. By more extensive oxidation, the binding site for bilirubin was lost. Photooxidation was accompanied by oligomerization of the albumin, as also observed by Vodrazka [25]. The present results indicate that this occurred independently of oxidation. In the light of this description of sensitized photooxidation of serum albumin it was investigated whether bilirubin, bound to the high-affinity site on the albumin molecule, could act as a sensitizer for oxidation of the protein. A singlet-oxygen-sensitizing effect of bilirubin has been demonstrated by McDonagh [2] and Foote [3], among others cited therein, who have studied the photochemical degradation of bilirubin in organic solvents. Related studies in aqueous systems [26] also point to an auto-sensitized photooxidation of bilirubin. In addition, Girotti [27] has suggested that bilirubin sensitizes photooxidation of red blood cell membranes. In contrast, we observed that albumin, isolated after irradiation in the presence of bilirubin, showed none of the properties of photooxidized albumin. This observation is compatible with suggestions of McDonagh [2] and Foote [3] that bilirubin in chloroform solution acts as a poor singletoxygen sensitizer but consumes singlet oxygen very effectively. On the other hand, bilirubin is photooxidized during irradiation of its complex with albumin. This process is preceded by a non-oxygen-consuming step, characterized by a small red shift and formation of a pigment which is more polar than bilirubin and is bound less firmly to albumin. These observations are consistent with those of Ostrow [26] who described an anaerobic photodegradation of bilirubin taking place only in the presence of albumin. Spectral evidence for the existence of a similar pigment during photolysis of the bilirubin . albumin complex has also been presented by Davies & Keohane [28]. It should be noted that this reaction is caused by an amount of light thousands of times less than the amount required for photooxidation of bilirubin. Practical interest in photooxidation of albumin and bilirubin is due to the extensive use of phototherapy in newborn infants with hyperbilirubinemia [4,5]. Bilirubin is toxic to various tissues and may cause lasting damage to the central nervous system, or death with yellow staining of basal ganglia of the brain (kernicterus). This may occur in the newborn, and especially in the prematurely born infant whose liver is incapable of excreting the pigment by the normal process of conjugation. In the healthy organism, the toxic effect of bilirubin is neutralized by binding to serum albumin. The binding may be critically reduced in the newborn, due to low concentration of albumin, or by competitive binding of fatty acids and drugs. Therapeutic measures include

Photooxidation of Albumin and Bilirubin

exchange transfusion or intensive irradiation with light during hours or days, reducing the concentration of bilirubin in the blood plasma. Serious, negative side effects have not been reported in the irradiated infants. However, Ode11 [6] has pointed out that photooxidized serum albumin has a decreased binding affinity for bilirubin and has discussed the possible significance of this in phototherapy. If bilirubin in vivo is capable of sensitizing oxidative processes, its carrier protein, serum albumin, should be especially exposed to oxidation. The presents results, however, indicate that photooxidation of serum albumin in vivo, sensititized by bilirubin, is unlikely. In agreement with this Cashore et al. [35] reported that the total bilirubin binding capacity of serum albumin did not decrease as a result of phototherapy in jaundiced infants. It has been generally assumed that bilirubin is photooxidized in the skin whereafter the oxidation products are excreted. Photooxidation of bilirubin in vitro has accordingly been used for assessment of the relative therapeutic efficiency of various light sources [29]. However, Ostrow [26] has found that increased amounts of unconjugated bilirubin appear in the bile of Gunn rats after irradiation with visible light. Similar observations have been made by Lund & Jacobsen in newborn infants after phototherapy 1301. These findings can hardly be explained by photooxidation of bilirubin. On the other hand, the above-described pigment, resulting from only a minute amount of irradiation of the albumin complex, may be the key to the mechanism of phototherapy on the following grounds. Bilirubin, as it occurs in the human body is predominantly the IX-a isomer 1311. The structure of this molecule has been elucidated by Bonnett et al. [32], using X-ray crystallography. They reported that bilirubin has the Z configuration at the C-4-C-5 and C-15-C-16 double bonds. A maximal array of intramolecular hydrogen bonds results in the apparently apolar character of bilirubin, which is soluble in chloroform and nearly insoluble in water at neutral pH. The Z configuration is a prerequisite for formation of these hydrogen bonds. Falk et al. [33] have demonstrated a photo-induced Z + E transition, i . e . cistrans isomerization, in a pyrromethenone. It may reasonably be suggested that a similar change of configuration occurs by irradiation of the bilirubin molecule which contains the same structure. The resulting pigment, bilirubin IX-a E, would be unable to form intramolecular hydrogen bonds and would accordingly appear more polar. This is in agreement with our finding of decreased chloroform extraction of bilirubin after the fast, non-oxygen-consuming photoprocess. Also the decreased binding to albumin is consistent with this idea. The E pigment would probably also be excreted in urine or bile without

221

A. 0 . Pedersen, F. Schi,%nheyder,and R . Brodersen

previous conjugation with glucuronic acid, since other water-soluble bilirubin isomers, IX-8, IX-y, and 1x4, are excreted in this way by virtue of their lack of intramolecular hydrogen bonds [31,34]. Excretion of these isomers by the Gunn rat which is unable to conjugate bilirubin, is likewise explained hereby. In the present state of knowledge it seems possible to hypothesize that phototherapy results in formation of a hydrophilic isomer of bilirubin, which is loosely bound to albumin and, at least partially, is excreted without previous conjugation. Photooxidation probably also takes place, although at a lower rate. We wish to acknowledge the helpful counsel of our colleagues and the excellent technical assistance of Nina Jdrgensen and Birthe Hust.

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A. 0. Pedersen, F. Schqhheyder, and R. Brodersen, lnstitut for Medicinsk Biokemi, Arhus Universitet, DK-8000 Arhus C, Denmark