Journal of Photochemistry and Photobiology B: Biology 61 (2001) 7886

www.elsevier.com / locate / jphotobiol

Fluorescence spectroscopy of normal mouse skin exposed to

5-aminolaevulinic acid and red light
Petras Juzenas
a

a,b ,

*, Vladimir Iani a , Saulius Bagdonas a,b , Ricardas Rotomskis b , Johan Moan a

Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
b
Laser Research Centre, Vilnius University, Sauletekio al. 9 c. 3, LT-2040 Vilnius, Lithuania
Received 28 June 2000; accepted 21 May 2001

Abstract
Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced
by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and
formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light
exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum
at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an
endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in
the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced
porphyrins are transported away from the treated area and partly deposited in remote skin sites. 2001 Elsevier Science B.V. All rights
reserved.
Keywords: Photodynamic therapy; Fluorescence spectroscopy; Photobleaching; Photoproduct; Protoporphyrin IX; Photoprotoporphyrin; Water-soluble
porphyrins

1. Introduction
Photosensitized reactions are widely applied in biology
and medicine, particularly for detection and treatment of
cancer. Photodynamic therapy (PDT) is a clinically applied
treatment modality of various malignant tumours based on
administration of an exogenous photosensitizer, which
selectively accumulates in tumours, followed by exposure
of the tumour area to light [1,2]. Photoactivation of the
photosensitizer induces cytotoxic reactions, which cause
tumour necrosis. Porphyrin-type dyes are widely used as
photosensitizers [3]. For the last decade the photosensitizer
protoporphyrin IX (PpIX) is being evaluated for use in
PDT. Accumulation of PpIX in tissues can be achieved by
administration (either systemically or topically) of its
natural precursor 5-aminolaevulinic acid (ALA) [4]. Kennedy and Pottier were first to apply ALA-based PDT
clinically [5].
In normal biosynthesis of haem ALA is produced in

*Corresponding author. Tel.: 147-22-934-263; fax: 147-22-934-270.

E-mail address: [email protected] (P. Juzenas).

mitochondria. The enzyme aminolaevulinate dehydratase

forms porphobilinogen (PBG) from ALA. Porphobilinogen
deaminase (PBGD) forms a tetrapyrrole ring from four
PBG molecules. Subsequently uroporphyrins (Up) are
formed. Decarboxylation then takes place and porphyrins
with decreasing polarity, such as hepta-, hexa-, pentacarboxylporphyrins and coproporphyrins (Cp), are formed.
PpIX is the last intermediate in this pathway. Finally, in
the mitochondria PpIX is converted into a haem by
ferrochelatase (FeC), an enzyme that inserts an iron atom
into PpIX. Haem has a negative feedback control on ALA
biosynthesis and photosensitizing concentrations of porphyrins never accumulate in tissues, except for the patients
with Erythropoietic protoporphyria (EPP) [6]. Exogenously administered ALA enters the haem biosynthesis pathway and bypasses this feedback control. Therefore, increased amounts of intermediate porphyrins are produced
in the cells. The FeC is not able to convert all of the
produced PpIX into a haem. Finally, PpIX accumulates in
tissues and photosensitization may occur. After application
of ALA preferential enrichment of porphyrins is usually
observed in tumours as compared to normal tissues. This
may be due to low activity of FeC [7] and probably also

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P. Juzenas et al. / Journal of Photochemistry and Photobiology B: Biology 61 (2001) 78 86

high activity of the rate-limiting enzyme PBGD in cancerous cells [8].

Phototransformations of porphyrins are observed under
light exposure in vitro and in vivo [913]. Such transformations are usually manifested as a decay (photobleaching) of both absorbance and fluorescence and a formation
of photoproducts. Inhoffen et al. were first to describe
photomodifications of PpIX, which, in many cases, are
transformations from a porphyrin-type to a chlorin-type
photosensitizer [14]. PpIX is one of the most photolabile
sensitizers. Phototransformations of ALA-induced PpIX
are observed in vitro and in vivo [1519] and may play a
clinical role [9,10]. Photobleaching of sensitizer may limit
efficacy of PDT and has to be taken into account when
choosing optimal light fluences and sensitizer concentrations [9,20]. On the other hand, photobleaching can
prevent photosensitization of normal skin adjacent to the
tumour area [9,21]. Photoproducts were found to have
photodynamic activity and may contribute to efficacy of
PDT [15,22,23].
The present work elucidates some important characteristics of photoproduct formation. Though several investigations deal with photobleaching and even identify some
photoproducts, the phototransformation processes, as well
as their influence on the PDT effect, are not yet fully
understood. Our findings obtained by fluorescence spectroscopy in vivo may contribute to the understanding of
these complicated photoinduced processes.

79

injection of hypnorm and dormicum (1:1, v / v) (Janssen,

Beerse, Belgium). Approximately 0.2 g of the freshly
prepared cream was applied on a single spot of approximately 1 cm 2 on each mouse and covered with adhesive
dressing (OpSite Flexigrid, Smith and Nephew Medical,
Hull, UK). Photobleaching was performed after 1314 h
of ALA cream application. This application time was
chosen because the content of PpIX itself in the skin did
not change significantly in this time interval [24]. The
adhesive dressing and the cream were removed before the
fluorescence measurements were carried out.

2.4. Porphyrin administration

Stock solutions of porphyrins (10 23 M of Cp, Up, PpIX
and PPp) were prepared by dissolving crude porphyrins in
ethylene glycol and NaOH (10 21 M). Before porphyrin
injection stock solutions were diluted in PBS to concentrations 10 24 M (pH 7.0) and 0.1 ml of such solutions
were injected subcutaneously into mice skin. Animals were
not anaesthetised during porphyrin injection and fluorescence measurements.

2.5. Light exposure

2. Materials and methods

Exposure of mouse skin to light was performed using a

tuneable Spectra-Physics 375 B dye laser (Spectra-Physics
Lasers, CA, USA) pumped by a Spectra-Physics 164 argon
ion laser. The fluence rate of laser light was 100 mW/ cm 2
at 635 nm (PpIX absorption maximum) on the surface of
the mouse skin.

2.1. Chemicals

2.6. Fluorescence measurements

ALA hydrochloride was obtained from PhotoCure

(Oslo, Norway). Crude ALA was dissolved in distilled
water (1:1, w / w) and solution was immediately used for
cream preparation. Protoporphyrin IX (PpIX) and photoprotoporphyrin (PPp) were purchased from Porphyrin
Products (Logan, UT, USA), and coproporphyrin III (Cp)
and uroporphyrin III (Up) from Nippon Oil (Tokyo,
Japan).

Fluorescence was measured by means of a Perkin Elmer

LS50B luminescence spectrometer (Norwalk, CT, USA).
Fluorescence spectra were recorded using a front-surface
set-up (Fig. 1), which gave a high-intensity fluorescence
signal necessary for detection of reliable spectra in vivo. In

2.2. Animals
Female hairless BALB / c mice were used. The animals
were normally active during the time of ALA application.
The mice were kept anaesthetised during the cream
application and fluorescence measurements.

2.3. ALA application

For topical application a cream was prepared using 20%
ALA w / w in an ointment (Unguentum, Merck, Darmstadt,
Germany). In order to facilitate proper application of the
cream all animals were anaesthetised with subcutaneous

Fig. 1. Experimental set-up for the front-surface measurements of

fluorescence in vivo.

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P. Juzenas et al. / Journal of Photochemistry and Photobiology B: Biology 61 (2001) 78 86

order to avoid any movements of the skin surface during

acquisition of fluorescence spectra each animal was anaesthetised and mechanically restrained with adhesive tape on
the sample holder of the luminescence spectrometer. A
thin glass plate was gently applied on the site of examination of the mouse skin in order to maintain skin surface flat
and parallel with the sample panel of the instrument. The
fluorescence excitation wavelength was set at 407 nm,
corresponding to the maximum of the Soret band of PpIX.
The excitation and emission slits were set at 5 and 10 nm,
respectively. Scattered excitation light was blocked from
the detected light with a 430 nm cut-off filter. Average
skin autofluorescence background measured on all animals
before ALA and porphyrin administration and exhibiting
monotonous decrease towards red spectral region without
any distinct peaks was subtracted from all fluorescence
spectra. All fluorescence spectra are corrected for the
spectral sensitivity of the luminescence spectrometer and
photomultiplier tube (PMT R928, Hamamatsu, Japan).
During photobleaching experiments fluorescence spectra
were acquired in the absence of laser exposure so that laser
emission did not interfere with the fluorescence signal. The
acquisition time (meaning the interruption between light
exposures) of each spectrum was ,1 min. Light exposure
did not cause any significant spectral changes in the
autofluorescence background of mouse skin.

3. Results

3.1. Heterogeneous photobleaching of fluorescence

After topical application of 20% ALA cream the mouse
skin exhibited red fluorescence with a spectrum typical for
PpIX, showing two characteristic peaks at 636 and 705 nm
(Fig. 2a, curve 1). Light exposure (100 mW/ cm 2 at 635
nm) caused bleaching and spectral changes of the fluorescence: A blue-shift of the main band ( lmax 5636628 nm)
and an appearance of new fluorescence bands at 588 and
675 nm were seen (Fig. 2b). Moreover, a broadening of the
main fluorescence band is distinct. Prior to light exposure,
the main fluorescence band had its maximum at lmax 5
636.060.2 nm with the Full Width at a Half Maximum
(FWHM) being 16.060.2 nm, while after an exposure of
2
40 min (fluence 240 J / cm ) the main fluorescence band
had its maximum at lmax 5628.561.4 nm with FWHM5
32.560.8 nm.

3.2. Formation of light-induced ( photo)products

Analysis of the fluorescence spectra was performed.
Prior to light exposure, the fluorescence spectrum (Fig. 2a,
curve 1) was identical with that of PpIX in cells in vitro
[25] and after subcutaneous injection of pure PpIX (data
not shown). Thus we assumed that primarily only PpIX
was present in the mouse skin after ALA application.

Fig. 2. (a) Fluorescence spectra of ALA-induced PpIX in normal mouse

skin recorded after laser light exposure (fluence rate 100 mW/ cm 2 at 635
nm). (b) The same spectra normalised at the main peak indicating spectral
changes induced by light exposure. Spectra from 1 to 16 represent
exposure times of 0, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12.5, 15, 20, 25, 30 and 40
min, respectively. Spectra represent averages from four mice.

Furthermore, the fluorescence spectra Fexp acquired after

light exposure were corrected for photobleaching of PpIX
(Fig. 3a) and the fluorescence spectra of light-induced
products F 9exp were obtained for given exposure times t exp
(Fig. 3b)
F 9exps l,t expd 5 Fexps l,t expd 2 FPpIXs l,t expd

(1)

where FPpIX is the fluorescence spectrum of PpIX corresponding to the exposure times t exp .0. Prior to light
exposure (t exp 50) no traces of fluorescence with characteristics similar to those of the light-induced products were
9 ( l,0)50.
seen and we assume that F exp
Calculations of the PpIX fluorescence spectra during
photobleaching FPpIX ( l,t exp ) (Fig. 3a) were performed
using the following procedure: The initial fluorescence
spectrum of PpIX (Fig. 2a, curve 1, t exp 50) was normalised at 636 nm to the fluorescence intensities at 636 nm of
the spectra recorded after light exposure (Fig. 2a, curves
216, t exp 50.540 min)
Fexps636,t expd
FPpIXs l,t expd 5 FPpIXs l,0d ? ]]]] ? k
FPpIXs636,0d

(2)

P. Juzenas et al. / Journal of Photochemistry and Photobiology B: Biology 61 (2001) 78 86

Fig. 3. Determination of the fluorescence spectrum of the light-induced

(photo)products. The illustration is presented for the case of the exposure
t exp 540 min. (a) Fluorescence spectrum (), recorded after exposure and PpIX fluorescence spectrum (- - -), defined from Eq. (2). Hence,
FPpIX ( l,40)5FPpIX ( l,0)30.053k, where k is assumed to be 0.9. (b)
Fluorescence spectrum after exposure corrected for PpIX photobleaching,
as defined from Eq. (1). Spectra represent averages from four mice.

where FPpIX ( l,0) is the initial fluorescence spectrum of

PpIX at t exp 50, FPpIX (636,0) is the PpIX fluorescence
intensity for l 5 636 nm at t exp 50 and Fexp (636,t exp ) is
the fluorescence intensity at l 5 636 nm measured after a
given exposure time t exp . The constant k50.9 was introduced to correct PpIX fluorescence in such a way that
negative fluorescence values are avoided around 640 nm in
the F 9exp spectrum after subtraction (Eq. (1)).
After light exposure the fluorescence spectrum corrected
for PpIX photobleaching exhibited three maxima with
peaks at 588.061.0, 623.260.8 and 674.860.2 nm (Fig.
3b).
For comparative analysis porphyrin fluorescence was
recorded in three different mice after subcutaneous injection of Cp, Up and PPp (Fig. 4). The simulated
photoproduct fluorescence spectrum calculated 2 h after
injection of Cp, Up and PPp exhibited maxima positioned
similarly to those seen after photobleaching of ALAinduced PpIX (Figs. 3b and 4b).
On the basis of data obtained in our model in vivo and
available from literature these peaks can probably be
assigned to the main fluorescence bands of an endogenous

81

Fig. 4. Simulation of the (photo)products spectra (solid upper line).

Spectra for Cp, Up and PPp were acquired in mouse skin 1 h (a) and 2 h
(b) after subcutaneous injection of porphyrins, as described in Materials
and methods. Note the appearance of the emission peak around 585 nm in
the case of Cp (b). Spectra represent averages from two mice.

metallo-porphyrin, presumably zinc-protoporphyrin (ZnPp)

( lmax 5588590 nm [26,27]), water-soluble porphyrin(s),
such as uroporphyrin (Up) ( lmax 5618620 [28,29]) and /
or coproporphyrin (Cp) ( lmax 5613 nm [28]) and the main
photoproduct, supposedly photoprotoporphyrin (PPp)
( lmax 5675 nm [17,25,29]).

3.3. Fluorescence kinetics

Fluorescence kinetics (Fig. 5) corresponding to the
fluorescence maxima of the PpIX and the light-induced
products were determined using Eqs. (1) and (2).
Prior to light exposure, ALA-induced PpIX in normal
mouse skin exhibited a high fluorescence intensity
(FPpIX (636,0)5640655 rel. u.). The PpIX fluorescence
FPpIX (636,t exp ), defined by Eq. (2), decayed rapidly during
light exposure (Fig. 5). Half of the PpIX content was
bleached already after an exposure of about 2 min (12
J / cm 2 ). Of the initial amount of PpIX 10% was left after
an exposure of about 12 min (72 J / cm 2 ) and ,5% was
left after the end of the exposure (40 min, 240 J / cm 2 ).
The PPp fluorescence F 9exp (675,t exp ), defined by Eq. (1),
rapidly increased in the beginning of the exposure and
reached a maximum after an exposure of about 5 min

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P. Juzenas et al. / Journal of Photochemistry and Photobiology B: Biology 61 (2001) 78 86

Fig. 5. PpIX ( lmax 5636 nm), PPp ( lmax 5675 nm) and Up / Cp ( lmax 5623 nm) fluorescence kinetics defined from Eqs. (1) and (2). Error bars represent
standard error within four mice.

(F 9exp (675,5)5104612 rel. u., 30 J / cm 2 ). About 65% of

the maximum value was reached after light exposure for
only 0.5 min (3 J / cm 2 ). The PPp fluorescence decayed
slowly under further exposure (Fig. 5). After prolonged
light exposure the PPp fluorescence Fexp (675,40)56768
rel. u. was found to be stronger than the PpIX fluorescence
FPpIX (636,40)52964 rel. u. (Fig. 5) under excitation at
407 nm.
The fluorescence kinetics at 623 nm were determined
using Eq. (1). This fluorescence is supposedly caused by
water-soluble porphyrins. It increased rapidly and reached
a plateau after light exposure of about 5 min (30 J / cm 2 ,
Fig. 5).
The fluorescence at 588 nm was relatively weak and
intensity variations were small (F 9exp (588,t exp )561 rel.
u., kinetic not shown).

3.4. Reappearance of porphyrin fluorescence

Fig. 6. (a) Reappearance of ALA-induced PpIX on the treated spot of

normal mouse skin the next day (19 h after treatment). The spectra
recorded before and immediately after light exposure are shown for
comparison (note the difference in fluorescence intensity, multiplication
factor is shown for each curve). (b) Localisation of ALA-induced
porphyrins at remote untreated (unexposed to ALA cream and laser
radiation) sites of normal mouse skin the next day after treatment. The
spectrum recorded immediately after treatment is shown for comparison.
Spectra represent averages from four mice.

The PpIX fluorescence in mouse skin was restored the

day after light treatment (Fig. 6a). The peak of the main
fluorescence band shifted back to near 636 nm. The
fluorescence peaks at 588 and 675 nm and broadening of
the main fluorescence band (620640 nm) were still
present (Fig. 6a). We also found the fluorescence of lightinduced (photo)products in the skin on remote and untreated sites (unexposed to ALA cream and light) of the
mice the day after light treatment (Fig. 6b).

4. Discussion
Light exposure of PpIX in living tissue leads to the
formation of singlet oxygen ( 1 O 2 ) and subsequently to

P. Juzenas et al. / Journal of Photochemistry and Photobiology B: Biology 61 (2001) 78 86

photooxidation products [1416]. 1 O 2 is generally thought

to be responsible for photosensitized cell killing. Furthermore, 1 O 2 is able to cause chemical modification of PDT
photosensitizers. Photochemical reactions initiated by 1 O 2
lead to destruction of porphyrin rings, accompanied by loss
of absorption and fluorescence (photobleaching). Chemical
modifications of the photosensitizer molecules may also be
induced without destruction of the main ring structure,
leading to formation of fluorescent chlorin-type photoproducts [12,13]. The PpIX photoproduct emitting fluorescence with a peak around 675 nm (Fig. 2) is attributed to
PPp [14,15,17]. This is also indicated by comparison with
the spectra of pure PPp in mouse skin (Fig. 4). Photoproducts with red-shifted absorption and fluorescence as
compared with initial porphyrins have been found to act as
photosensitizers [15,22,23]. Thus, PPp may contribute to
the efficacy of ALAPDT. Moreover, we observed that
after prolonged light exposure the intensity of PPp fluorescence was stronger than that of PpIX (Fig. 5). Earlier we
have found similar PPp formation kinetics in cells incubated with ALA in vitro and exposed to 635 nm laser
radiation [25].
Spectral changes, such as blue-shift and broadening of
the fluorescence band, are caused by processes other than
simple photobleaching. Spectral changes, similar to some
of those seen during light exposure, may be related to
movement of the fluorophore to other localisation sites
[10]. Light exposure destroys binding sites and releases
PpIX molecules, which can then move to other binding
sites and structures, thus changing the targets for PDT and
contributing to photodamage of the cells [6,30]. It is well
known that the shape of the spectra of porphyrins and
chlorins are dependent on the environment (protein binding, pH, etc.) [31]. Different photodegradation rates appear
to be related to different types of binding sites [30,32]. All
these factors may contribute to the spectral changes of
PpIX fluorescence during light exposure.
In the pathway of haem biosynthesis, all intermediate
porphyrins formed from ALA are finally converted to
haem. The hydrophilic character of the intermediate watersoluble porphyrins should lead to a rapid clearance from
tissues [33,34]. Consequently, endogenous ALA, as well as
exogenously applied ALA, should gradually cause accumulation of only PpIX. However, we found a lightinduced increase of fluorescence around 580625 nm
(Figs. 2b, 3 and 5). This may indicate that light interferes
with the haem biosynthetic pathway and leads to formation
of water-soluble porphyrins. An inverse correlation between the formation of PpIX and water-soluble porphyrins
has been found in vitro. Exposure of a cell suspension to
light led to an increased fluorescence at 616 and 678 nm
[28]. This supports the hypothesis of light-induced accumulation of water-soluble porphyrins. Sensitisation of
PpIX in cells may lead to a reduction of mitochondrial
viability, which may prevent further formation of PpIX
resulting in the blockage of the biosynthetic pathway.

83

Thus, water-soluble porphyrins may accumulate. Watersoluble porphyrins with fluorescence around 620 nm have
been observed in mouse tumours in vivo, but, contrary to
our findings, not in normal skin [29].
Changes of parameters other than light exposure, such
as temperature, glucose concentration and incubation time,
influence the relative amount of ALA-induced water-soluble porphyrins compared with PpIX [28,29]. Water-soluble
porphyrins are generally thought to be responsible for
photodynamic damage to the vascular system of tumours
[35]. Skin photodamage sensitised by water-soluble porphyrins is likely to be similar to the damage seen in
patients with the disease porphyria cutanea tarda [36].
Vessel damage and damage originating from lysosomal
rupture are likely to occur, while PpIX-sensitised skin
damage is differently manifested and, probably, originates
from mitochondrial and plasma membrane damage [36].
Hydrophilic porphyrins Up and Cp were found to be much
less dark cytotoxic than lipophilic PpIX after incubation in
cells in vitro [37]. However, the photodynamic efficacy for
cell phototoxicity and vascular injury was recently reported
to be as Up,Cp,PpIX [37]. Recently Finlay et al.
observed an irradiance dependent appearance of an emission band with a peak around 620 nm in vivo, which was
adequately fitted with the weighted sum of the fluorescence
spectra of Cp and Up in lipid solutions in vitro [38]. In our
model the emission spectra of the photoproducts were
simulated using those obtained in vivo, i.e. after subcutaneous injection of porphyrins in mouse skin. The similarity
of the spectra is reasonably apparent despite of the slight
blue-shift in the spectra obtained after injection of porphyrins compared with that obtained after topical application of ALA (Figs. 3b and 4b), which can be explained by
the different localisation of porphyrins after two different
administration modalities. Naturally, the relative weight of
different porphyrins in the complex spectrum should be
considered before the adequate fitting is made. The maximal fluorescence intensity of water-soluble porphyrin(s) in
normal mouse skin was found to be about 20 times lower
than the initial PpIX fluorescence intensity, while these
fluorescence intensities were found to be similar at the end
of the light exposure (Fig. 5). Thus, water-soluble porphyrins may certainly contribute to the tissue-damaging
effect during ALAPDT. It is also possible that PDTinduced vascular damage contributes to the accumulation
of water-soluble porphyrins and metallo-porphyrins.
After light exposure, we observed increased fluorescence around 588 nm (Figs. 2b and 3). This may indicate
the formation of metallo-porphyrins. Zinc-protoporphyrin
(ZnPp) emission around 590 nm has been observed in
extracted liver cells and blood erythrocytes in vitro
[26,27]. Though we have not employed ZnPp in our
comparative study, we observed the appearance of the
emission peak around 585 nm 2 h after injection of Cp
(Fig. 4b). The ZnPp is a normal metabolite that accumulates in trace amounts in erythrocytes during haemoglobin

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P. Juzenas et al. / Journal of Photochemistry and Photobiology B: Biology 61 (2001) 78 86

synthesis. Both iron and zinc are ferrochelatase substrates

and, consequently, excess ZnPp may be formed as a
by-product of the haem biosynthesis [39]. Contrary to
haem (ferric protoporphyrin), ZnPp exhibits significant
fluorescence [40] and, therefore, may become discernible
when PpIX is photobleached. Enhanced emission around
550600 nm has been observed in mouse tumours [29],
but not in cells in vitro [25,28]. Bottiroli et al. observed a
presence of 580 nm-emitting species of haematoporphyrin
derivative (HpD) in solutions and in cells in vitro and
suggested that this could be a consequence of modifications in HpD induced by the different cellular microenvironments [41]. Andreoni et al. attributed the blueshifted emission of aging solutions of hematoporphyrin
and its derivatives to the formation of aggregated polymeric structures of porphyrins [42]. We have earlier
demonstrated that metallo-porphyrins exhibiting fluorescence around 580 nm are easily formed when solutions of
free porphyrins are kept in laboratory glassware containing
trace amounts of metallic ions [43]. Metallo-porphyrins
probably do not play any important role during PDT, since
they show a low photosensitizing activity [44] and are
present only in trace amounts in the skin (Figs. 2 and 3).
Photobleaching of ALA-induced PpIX fluorescence in
animal tissues and in humans has been reported by several
investigators [19,45]. Clinically relevant light fluences
(40200 J / cm 2 ) cause degradation of 7095% of the PpIX
initially present [30,45]. In our model, 95% of PpIX was
photobleached in normal mouse skin by a fluence of
180240 J / cm 2 (Fig. 5). Photobleaching of PpIX may
limit the therapeutic effect, which may be a disadvantage
whenever a strong PDT effect is required. However,
photobleaching can prevent sensitisation of normal skin
adjacent to the tumour area and enhance PDT selectivity
[9,21].
The PpIX fluorescence in mouse skin was restored the
day after light treatment. The peak of the main fluorescence band shifted back to near 636 nm (Fig. 6a). This
most probably shows that PpIX is resynthesised from
ALA, which is still present in the skin even 20 h after
treatment (totally 34 h after cream had been applied). If so,
the light exposures employed here have left the biosynthetic machinery of PpIX synthesis relatively undamaged. However, it is possible that reappearance of PpIX
takes place in the treatment spot from adjacent untreated
tissue and / or blood circulation, since prolonged topical
application of ALA induces a systemic effect in mice (Fig.
6b and [24]). This may be explained by the fact that we
employed a long incubation time (13 h) and a high ALA
concentration (0.2 g / cm 2 of 20% ALA cream). Clearly,
the peaks at 588 and 675 nm, as well as the shoulder
around 620 nm, are still present and indicate that lightinduced (photo)products have not been eliminated at this
late time (Fig. 6a). This argues for the significance of
damage to the microvascular system. Light-induced
(photo)products seem to be transported away from the
treated spot and partly deposited in remote tissues (Fig.

6b). Van der Veen et al. observed reappearance of ALAinduced PpIX fluorescence 12 h following the light
treatment [45]. They found no evidence that ALA was
available in excess at 4 and 10 h after topical application
and suggested that the increase of PpIX after photobleaching may result from decreased metabolisation of PpIX to
haem after light treatment [45]. Unfortunately, sufficient
knowledge is unavailable now concerning the detailed
pharmacokinetics of exogenously applied ALA and the
relationship between the pharmacokinetics of ALA and
that of ALA-induced PpIX in tissues and plasma. The
appearance of water-soluble porphyrins and photoproducts
and reappearance of PpIX fluorescence after light exposure
may also contribute to higher efficacy of ALAPDT using
light dose fractionation [46].
In conclusion, PpIX photobleaching and photoproduct
formation in vivo seem to be accompanied by disturbances
of the haem biosynthetic pathway. Light exposure of ALAtreated skin leads to enhanced production or retention of
water-soluble porphyrins, such as Up and / or Cp. Vascular
damage may play a role. After light exposure reappearance
of PpIX is observed in the light-exposed skin spot.
Photobleaching, appearance of water-soluble porphyrins
and formation of a chlorin-type photoproduct during light
exposure may modulate the efficacy of ALAPDT. Furthermore, a broad-band exposure, covering not only the
absorption of PpIX, but also those of water-soluble porphyrins and photoproducts, seems to be advisable for
ALAPDT. Further investigations are of great importance
to study the identification and formation mechanisms of
light-induced (photo)products.

5. Abbreviations
PDT
ALA
PpIX
Up
Cp
PPp
FWHM
F
rel. u.

Photodynamic therapy
5-Aminolaevulinic acid
Protoporphyrin IX
Uroporphyrin
Coproporphyrin
Photoprotoporphyrin
Full width at a half maximum
Fluorescence intensity
Relative fluorescence units

Acknowledgements
The present work was supported by The Research
Council of Norway, by The Norwegian Radium Hospital
Research Foundation and partially by the Lithuanian State
Science and Studies Foundation.

References
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