Photochemistry of bacteriochlorophylls in human blood plasma: 2. Reaction mechanism investigated by product analysis and deuterium isotope effect

Article abstract of DOI:10.1111/j.1751-1097.2009.00678.x

Transmetalated (Pd) bacteriochlorophyll derivatives are currently being clinically tested as sensitizers for photodynamic therapy. Protocols using short delay times between injection and irradiation generate interest in the photochemistry of these pigments in the blood. Using near-infrared irradiation where these pigments absorb strongly, we have studied the mechanism of photo-oxidation in two lipoprotein fractions, low- and high-density lipoproteins, derived from human blood plasma that preferentially accumulate these pigments (Dandler et al. [2009] Photochem. Photobiol., 85, in press). Using quenchers of reactive oxygen species, and chemical reporters, in particular peroxides generated from cholesterol as an inherent component of the lipoproteins, a Type II mechanism generating singlet oxygen has been demonstrated for Pd- and Zn-bacteriopheophorbides. In homogeneous systems, accelerated bleaching in D₂O, compared with H₂O, supports this mechanism. An unusual deuterium isotope effect was observed, by contrast, in heterogeneous amphiphilic-water systems. In the early phase, and under high oxygen concentrations, again a positive D-isotope effect is observed which later, in a second phase, is reversed to a negative D-isotope effect. The latter cannot be explained by heterogeneous pigment populations in the amphiphilic system; we, therefore, conclude a mechanistic switch, and discuss a possible mechanism.

Full text of DOI:10.1111/j.1751-1097.2009.00678.x

Photochemistry and Photobiology, 2010, 86: 342–352
Photochemistry of Bacteriochlorophylls in Human Blood Plasma:
2. Reaction Mechanism Investigated by Product Analysis and
Deuterium Isotope Effect
J o¨ rg Dandler, Brigitte Wilhelm and Hugo Scheer*
Department Biologie I - Botanik, Universit a¨ t M u¨ nchen, Munich, Germany
Received 17 July 2009, accepted 27 October 2009, DOI: 10.1111 ⁄ j.1751-1097.2009.00678.x
(
15,16), the photolability of many such derivatives (11,12,17)
ABSTRACT
and possibly dark degradations including oxidative ring
opening (18,19). The effectiveness, in spite of the rapid
clearance, supports evidence for indirect modes of action in
PDT not involving a direct attack on the target cells; these
indirect modes include apoptotic signaling by ROS and a
vascular action (20–27). Pigments with rapid turnover times,
like BChl derivatives, are particularly suited for vascular
targeting. To design treatment protocols appropriate for this
rapid clearance, the pharmacokinetics of photosensitizers
becomes of considerable practical interest. This includes, in
particular, their photochemistry in the blood where at least
part of the primary action will take place when protocols are
used involving short delay periods between injection and
irradiation, or between subsequent irradiations (28,29).
Mechanistic details of the photodynamic action are cur-
Transmetalated (Pd) bacteriochlorophyll derivatives are cur-
rently being clinically tested as sensitizers for photodynamic
therapy. Protocols using short delay times between injection and
irradiation generate interest in the photochemistry of these
pigments in the blood. Using near-infrared irradiation where
these pigments absorb strongly, we have studied the mechanism of
photo-oxidation in two lipoprotein fractions, low- and high-
density lipoproteins, derived from human blood plasma that
preferentially accumulate these pigments (Dandler et al. [2009]
Photochem. Photobiol., 85, in press). Using quenchers of reactive
oxygen species, and chemical reporters, in particular peroxides
generated from cholesterol as an inherent component of the
lipoproteins, a Type II mechanism generating singlet oxygen has
been demonstrated for Pd- and Zn-bacteriopheophorbides. In
1
homogeneous systems, accelerated bleaching in D O, compared
2
rently only partly understood. Singlet oxygen in its D state
g
1
2
with H O, supports this mechanism. An unusual deuterium
(
O ) has been shown to be a major ROS in PDT, not only as a
2
isotope effect was observed, by contrast, in heterogeneous
amphiphilic-water systems. In the early phase, and under high
oxygen concentrations, again a positive D-isotope effect is
observed which later, in a second phase, is reversed to a negative
D-isotope effect. The latter cannot be explained by heterogeneous
pigment populations in the amphiphilic system; we, therefore,
conclude a mechanistic switch, and discuss a possible mechanism.
necrotic agent but also as a signal, for example, for apoptosis
22,23,30,31). It can be identified by chemical and physical
characteristics such as the photo-oxidation products of cho-
(
lesterol (8,32), b-carotene (33) or limonene (34), the phospho-
rescence at 1270 nm (35) or the pronounced H ⁄ D isotope
1
effect on the lifetime of
pronounced in water, where replacement of H O with D O
2
O (36). The latter is particularly
2
2
1
2
increases the lifetime of O by an order of magnitude (37,38).
While there is much research into the photochemistry of
BChl related to photosynthesis (39,40), little is known about
their photochemistry in blood. We have recently shown (41)
that most of these pigments when added to blood are present
in the blood plasma (BP) fraction, mainly in the low- (LDL)
and high-density lipoproteins (HDL); the highly-polar tauri-
nated Pd-complex, WST11 (structure see Fig. 1), accumulates
also in the high-density protein (HDP) fraction (41). The better
solubility in water is accompanied by a decreased photosta-
bility; it is, therefore, of interest if these two properties are
linked. As it has been recognized that the generation of ROS
species by these pigments depends on their microenvironment
(42), we studied the photochemistry of three representative
sensitizers in these BP fractions, namely WST11, and the
Pd- and Zn-complexes of bacteriopheophorbide (BPheid),
namely Pd-BPheid (WST09) and Zn-BPheid (for structures,
see Fig. 1). Here, we report the photosensitization mechanism
of the pigments in the different BP fractions, using chemical
quenchers, product analysis of cholesterol, which is an
INTRODUCTION
Photodynamic therapy (PDT) uses reactive oxygen species
(
ROS) to treat malignancies including cancer. ROS are
generated by irradiating photosensitizing dyes with visible
VIS) or near-infrared (NIR) light. Pigments absorbing
(
strongly in the NIR are particularly advantageous because
tissue is relatively transparent to NIR light (1). Bacteriochlo-
rophyll (BChl) derivatives are among such sensitizers (2–10).
They show some favorable properties including low dark
toxicity and relatively high accumulation in transformed
tissue. In particular, Pd derivatives are chemically more stable
(
11,12) and give higher quantum yields for triplet generation
than the parent Mg-complexes (13,14). The pigments are
rapidly cleared from the body, which is probably due to a
combination of factors including an active excretion system
*
ꢀ
Corresponding author email: hugo.scheer@lmu.de (Hugo Scheer)
2010 The Authors. JournalCompilation. The AmericanSocietyof Photobiology 0031-8655/10
3
42

Photochemistry and Photobiology, 2010, 86 343
(
1
Horiba Jobin Yvon, Munich, Germany) equipped with an SVX ⁄ LAX
¨
450 lamp (Muller Elektronik-Optik, Moosinning, Germany) and
R374E photomultiplier (Hamamatsu Photonics, Hamamatsu City,
Japan), controlled by Quantus (A. Pazur, Department Biology
I—Botany, LMU Munchen, Munich, Germany).
¨
Sample preparation. Dialyzed fractions of human BP, loaded with
various tetrapyrroles, were obtained by sequential density gradient
ultracentrifugation (SDGU) as described previously (41). CES solu-
tions were prepared by mixing a pigment solution in CrEL ⁄ ethanol
(
1:1 vol ⁄ vol) with H
were concentrated with Centricon YM-30 centrifugal filter devices
Millipore, Schwalbach, Germany). For H ⁄ D-exchange of HDL
samples, pigment-loaded HDL were diluted with HDL samples not
containing pigments to an optical density of 1.0 at the Q absorption
maximum and dialyzed overnight at 4ꢂC against H O or D O under
otherwise identical conditions (Servapor tubing, MWCO: 12,000–
4,000; Ø: 16 mm; Serva, Heidelberg, Germany). Micellar solutions in
CrEL were obtained by mixing a solution of WST09 (17.9 lM,
resulting in an optical density of 1.0 at the Q absorption maximum of
2
O in a 1:19 (vol ⁄ vol) ratio. If necessary, samples
ꢁ
(
y
2
2
ꢁ
1
y
Figure 1. Molecular structures and abbreviations of pigments. Struc-
tures are numbered according to IUPAC-IUB (91). Pigments with an
WST09) in CrEL ⁄ ethanol (1:1 vol ⁄ vol) with either H
2 2
O or D O in a
2
isocyclic ring are generally present as 13 -epimer mixtures, but only the
1:39 (vol ⁄ vol) ratio.
Irradiations. Samples (100 lL) were irradiated in an ultra-micro cell
2
3 R-epimers are shown as they are usually present in large excess over
1
1
(
¨
105.202-QS; Hellma, Mullheim, Germany) from the top using a cold-
2
3 S-epimers. The respective chlorins generated by oxidation have a
light source (KL 1500 electronic; Schott, Mainz, Germany) equipped
with either a RG630 or a RG695 low-pass filter (Schott). The photon
flux at the position of the cuvette was determined with a photometer
consisting of a SKP 200 display meter and a SKP 218 ⁄ 730 FR far-red
sensor (Skye Instruments, Llandrindod Wells, UK); the NIR light
D7,8 double bond while the respective porphyrins also have a D17,18
double bond.
)
2 )1
inherent chemical reporter of lipoproteins, and deuterium
isotope effects as mechanistic probes. A separate report (43)
focuses on: the photostability of the pigments in the different
BP fractions; the oxygen consumption during irradiation; the
formation of hydro- and endoperoxides; and the light-induced
cross-linking of BP proteins.
intensity was maximally 530 lE m
s
at 730 nm (30 nm full width
at half height). The absorption changes of the sample during
irradiation were monitored with a DAD system. The samples were
not stirred.
Oxygen determination. Oxygen consumption was measured with a
Clark-type electrode consisting of a DW1 electrode chamber and an
Oxylab control unit, controlled by Oxylab 1.10 (all Hansatech,
Pentney, UK). The electrode was calibrated according to the manu-
facturer’s instructions. The sample (1.0 mL) was kept at 25ꢂC and
stirred with a power setting of the controller of 30%.
Extraction of tetrapyrroles and carotenoids. A lipoprotein sample
(1 mL) was mixed (30 s vortexing) with 1 mL ice-cold ethanol. This
mixture was mixed by vortexing (30 s) with 5 mL methylene chloride,
and then centrifuged (1000 g, 3 min). The lower methylene chloride
phase was completely removed and, after drying by rotary evapora-
tion, the residue was extracted with acetone; the remaining solid on the
wall of the flask (mainly insoluble lipids) should be colorless. The
acetone solution was dried in a rotary evaporator, and the sample
stored under argon at )20ꢂC until HPLC analysis.
HPLC analysis. The HPLC system used consisted of a 600E
multisolvent delivery system (Waters, Eschborn, Germany), a GROM-
SIL 120 ODS-5 ST (5 lm) column (0.4 · 30 cm) (Alltech Grom,
Rottenburg-Hailfingen, Germany) and a DAD system (see above). The
MATERIALS AND METHODS
All experiments were carried out under green light (<1 lE m)2 s)1
and at ambient temperature unless stated otherwise.
)
Materials. Chemicals were analytical grade or better. Unless
otherwise noted, they were obtained from Sigma-Aldrich (Taufkir-
chen, Germany), Roth (Karlsruhe, Germany) or Merck (Darmstadt,
ꢁ
Germany). Cremophor EL (CrEL) was from Fluka (Taufkirchen,
2
Germany) and D O (99.6% D) from Merck. Diethyl ether was dried
and peroxides removed using Alumina B-Super I columns (ICN
Biomedicals, Eschwege, Germany). Doubly demineralized water was
used for all aqueous solutions. Blood products were obtained from the
Bavarian Red Cross (Munich, Germany) and stored at 4ꢂC for up to
ꢁ
4
weeks. CE is a 1:1 (vol ⁄ vol) mixture of Cremophor EL (CrEL) and
ethanol, CES a 1:9 (vol ⁄ vol) mixture of CE and normal saline.
Pigments. All preparations were carried out under green light
2
binary system was composed of solvent A: 70% acetone, 30% H O
(pH 3.5 with acetic acid); and solvent B: 100% acetone; the flow rate
was 1.2 mL min ; using the following gradient: 0 min: 100% A;
<1 lE m)
and during use. Reactions were followed by absorption spectroscopy.
2
s
)1
) and argon. Solvents were gassed with argon prior to
)1
(
4 min: 100% A; 19 min: 0% A; 20 min: 0% A; 24 min: 100% A;
30 min: 100% A. Chromatograms were extracted at 430 nm from the
3D data sets. All solvents were degassed before use. Tetrapyrroles were
identified by co-chromatography with authentic samples and also by
their absorption spectra. Carotenoids were identified by comparison
with published HPLC analyses of BP carotenoids and ⁄ or by their
absorption spectra (44–46).
HPTLC of cholesterol oxidation products. Lipid extraction: Lipids
were extracted from the irradiated samples (0.5 mL) with a mixture of
methylene chloride and methanol (1:1 vol ⁄ vol) (1 mL). The methylene
chloride phase was concentrated to 50 lL under a stream of argon,
and diluted with 50 lL of methanol. If reduced peroxides were
Pigments were stored dry, in the dark and under argon at )20ꢂC. Pd-
bacteriopheophorbide (Pd-BPheid; WST09; Tookad ) was a gift from
ꢁ
Negma-Lerads (Magny-Les-Hameaux, France). Zn-bacteriopheophor-
bide (Zn-BPheid) was prepared as previously described (41). Palladium
1
1
-oxo-15-methoxycarbonylmethyl-rhodobacteriochlorin 13 -(2-sulfo-
3
ꢁ
ethyl) amide dipotassium salt (WST11; Stakel , Steba Biotech,
Toussus-Le-Noble, France) was provided by A. Scherz (Weizmann
Institute of Science, Rehovot, Israel). Synthetic all-trans-b-carotene
was from Sigma (No. C-9750; Taufkirchen, Germany).
Spectroscopy. Static absorption spectra were recorded with a UV
401 PC Spectrometer equipped with an ISR 240-A Integrating Sphere
2
Assembly, controlled by Hyper UV 1.5 (all Shimadzu, Duisburg,
Germany) based on Spectacle (LabControl, Cologne, Germany).
Dynamic absorption spectra were recorded with a diode array
detection (DAD) system consisting of a Tidas UVNIR ⁄ 16–1024 ⁄
4
assayed, a methanolic solution of NaBH (50 mM) and NaOH (10 mM)
was used for dilution, and the samples were incubated for 1 h at
ambient temperature. In both cases, an aliquot (16 lL) was applied to
HPTLC plates (Kieselgel 60 on aluminum sheets; Merck), which were
developed with a mixture of n-hexane and ethyl acetate (1:1 vol ⁄ vol) in
1
00-1 diode array detector and a CLH 20 W lamp controlled by
Spectralys 1.82 (all J&M, Aalen, Germany) based on Spectacle
a
horizontal chamber. After development, hydroperoxides were
stained dark blue within a few minutes at ambient temperature with
N,N,N¢,N¢-tetramethyl-p-phenylenediamine (TMPD, 6 mM) in H O,
(
(
LabControl). Fluorescence spectra and relative fluorescence units
RFU) were measured with a Spex Fluorolog 212 spectrofluorometer
2

3
44 J o¨ rg Dandler et al.
methanol and acetic acid (50:50:1 vol ⁄ vol ⁄ vol, modified from Ref.
47]). Lipids were stained with CuSO (0.32 M) in H O, concentrated
PO
Upon heating the wet plates with a heat gun, the lipid spots gave
characteristic colors depending on their structures: Chol, purple;
Zn-BPheid, and only in HDL (for supporting information, see
Table S1). Again, the photostability was not increased by
quenchers for Type I reactions (sucrose, glycerol, DMSO). The
response to quenchers for Type II reactions was heteroge-
[
H
4
2
3
4
and methanol (85:10:5 vol ⁄ vol ⁄ vol, modified from Ref. [48]).
(O)OH-Chol, light blue; 7-keto-Chol, yellow; sodium cholate, olive;
most other lipids, brown.
Cholesterol esterase reaction: 2.5 mg cholesterol esterase (Porcine
pancreatic CholEase; 33.1 U mg ; Fluka, Taufkirchen, Germany)
and 20 mg sodium cholate were dissolved in 15 mL phosphate buffer
neous. D
H
2
O, which enhances the lifetime 10-fold relative to
2
O (36), slightly increased photostability. Sodium azide was
ineffective, probably because of its ionic nature; like many
other ROS quenchers, it is too polar to interact with
membranes (55) and, therefore, with membrane-bound pig-
ments. As quenching requires physical contact with the ROS
)
1
(50 mM; pH 7.2). For each assay 400 lL of this solution and 100 lL
of the respective sample were incubated for 1 h at 37ꢂC, and
subsequently analyzed by HPTLC (see above). The enzymatic activity
(
55), 1,4-diazabicyclo[2.2.2]octane and b-carotene were tested
1
was checked by
-propanol.
Preparation of standards: Primary and secondary cholesterol
a
solution of cholesterol oleate (12.3 mM) in
2
as lipophilic O quenchers. The former only slightly increased
2
photostability while the latter was more effective even consid-
ering that CrEL, used for solubilizing b-carotene, was partly
responsible for this protection. As the natural carotenoid
concentration in human BP is only about 2 lM (56), it would
only marginally protect pigments (and other molecules) from
this level of ROS attack. Among the other 19 standard amino
acids (see Table S1), photostability was also increased by
oxidation products were identified by co-chromatography against
freshly prepared standards (49,50). A mixture of 3b-hydroxycholest-
5-ene-7a-hydroperoxide (7a-OOH-Chol) and 3b-hydroxycholest-5-
ene-7b-hydroperoxide (7b-OOH-Chol) was produced by overnight
incubation of an LDL or HDL lipoprotein fraction with the same
volume of aqueous CuSO (20 mM) and citric acid (50 mM; adjusted to
4
pH 4.0 with NaOH) at 37ꢂC. A mixture of the respective alcohols
(
(
(
7-OH-Chol) was produced by treatment of 100 lL 7-keto-cholesterol
12.5 mM) in methanol with 100 lL of a methanolic solution of NaBH
50 mM) and NaOH (10 mM). The solvent was evaporated with a
cysteine, methionine, tyrosine and tryptophan that react with
1
4
O
2
(57,58) and, therefore, are quenchers of Type II reactions
52). Proline had no significant effect, although it can quench
(
1
stream of argon, and the residue containing 7ab-OH-Chol was
dissolved in a mixture of methylene chloride and methanol (1:1
vol ⁄ vol). 3b-hydroxy-5a-cholest-6-ene-5-hydroperoxide (5a-OOH-
Chol) was produced by irradiating a mixture of 100 lL Chol
O under certain conditions (59).
2
(12.9 mM) in methanol with 100 lL rose bengal (50 mM) in methanol
Cholesterol oxidation products
for 1 h with white light from a cold-light source (see above) at
maximum light intensity. The respective alcohol (5a-OH-Chol) was
produced by chemical reduction of 5a-OOH-Chol with NaBH in
4
methanol, under the conditions used for the reduction of 7-keto-Chol
to 7-OH-Chol (see above).
A more rigorous approach to distinguish photo-oxidation
mechanisms was the analysis of oxidation products of choles-
terol (Chol) and its esters (CholE) (60) by HPTLC, either
directly using TMPD staining, or indirectly, after reduction
with sodium borohydride to the corresponding more stable
hydroxyl derivatives, using acidic CuSO4 staining. Type I
reactions generate 7-OOH-Chol (Fig. 2), predominantly in the
7b-configuration, together with traces of 7-OH-Chol, 5,6-
epoxy-Chol and 7-keto-Chol. Type II reactions generate
mainly 5a-OOH-Chol, accompanied by traces of 6-OOH-Chol
RESULTS
Effects of ROS quenchers
Type I reactions involving radicals and Type II reactions
1
initiated by singlet oxygen ( O
2
) can be distinguished by
(
both epimers).
Lipids extracted from nonirradiated HDL contained no
quenchers (see Bonnett [8] for definition). For initial tests, D-
mannitol (51) was used as a quencher for Type I reactions and
L-histidine (52) for Type II reactions while sodium ascorbate
peroxides; those from LDL contained trace amounts of several
peroxides (data not shown). In all irradiated samples, 5a-
OOH-Chol was found (see supplementary material, Fig. S1).
The majority of the peroxides, however, were derivatives of
different CholE that could not be separated using the HPTLC
system that was optimized for analysis of Chol peroxides. We
found, however, that the CholE peroxides can be hydrolyzed
nearly quantitatively (compare Fig. 3a and Fig. S1) by cho-
lesterol esterase (CholEase) to form the corresponding Chol
peroxides, yielding 5a-OOH-Chol as the main oxidation
(
53,54) was used as a nondiscriminating antioxidant. The
photostability of the sensitizer, Zn-BPheid, was not increased
by the presence of mannitol, but was enhanced by histidine
and, even more so, by ascorbate, indicating that photobleach-
1
ing is due to O . The same is true for Pd-BPheid: although it
2
bleached much more slowly (43), the effects of the quenchers
were comparable. There was also no significant difference
between LDL and HDL. Experiments with a larger set of ROS
quenchers were undertaken with the more rapidly bleaching
Figure 2. Structures of cholesterol (left) and its main oxidation products generated by Type I (top right) and Type II photoreactions (bottom right).
Alcohols are obtained from the respective peroxides by reduction with sodium borohydride.

Photochemistry and Photobiology, 2010, 86 345
product (Fig. 3a). In addition to traces of nonhydrolyzed
CholE (peroxides), two further peroxide bands of unknown
nature were detected in each sample. These may be peroxides
derived from fatty acids, but they were not identified.
Phospholipid peroxides remain at the origin in the chroma-
tography system used (50). In the context of investigating the
photo-oxidation mechanism, it was important that they
contained no 7-OOH-Chol as shown by co-chromatography
of the corresponding alcohols (see below) against an authentic
sample obtained by reduction of 7-keto-Chol. The unknown
product II is formed in small amounts only in LDL, but
approaches or even exceeds 5a-OOH-Chol in HDL, irrespec-
tive of the sensitizer used. Using the summed intensities of
the stained peroxide bands as an estimate for peroxide
generation, their concentrations decreased in the following
order: LDL + Pd-BPheid >> HDL + Pd-BPheid @ LDL +
Zn-BPheid > HDL + Zn-BPheid. Although this is only a
rough estimate, it conforms with the results from a quantita-
tive test assay based on the formation of 2¢,7¢-dichloro-
fluorescein (43).
Chol) can be clearly identified (61). Furthermore, staining with
4
CuSO permits a clear distinction between hydroxylated Chol
derivatives by their blue color from other Chol derivatives. The
results are compatible with a Type II reaction: after reduction,
all four irradiated samples contained 5a-OH-Chol, the largest
amount being present in LDL loaded with Pd-BPheid
(Fig. 3b). There is also an unidentified band with the r value
f
of 7b-OH-Chol (assignment of the two 7-OH-Chol epimers by
their relative r values [49,50]), but it lacks the characteristic
f
light blue color that is clearly visible in the corresponding
standard. Furthermore, in no case was there an accompanying
band of 7a-OH-Chol that would have also been generated by a
Type I reaction.
The Type II mechanism also dominates reactions of Pd-
BPheid in many organic solvents. If the pigment was irradiated
together with Chol, 5a-OOH-Chol (5a-OH-Chol after reduc-
tion) was formed in most solvents, and 7-OOH-Chol (7-OH-
Chol after reduction) was never detected (see Figs. S3 and S4).
In some cases, the oxidation products apparently reacted with
the solvent, thereby forming nonassignable Chol derivatives.
HPTLC of the peroxides gives only poor separations of the
epimers of 7-OOH-Chol and 5a-OOH-Chol (see supplemen-
tary material, Fig. S2). The resolution of the HPTLC system
and the stability of the products could be considerably
No oxidation products were found in DMSO, which is not only
(63,64).
1
a quencher of hydroxyl radicals (62), but also of O
2
Carotenoid oxidation products
4
improved if the peroxides were reduced with NaBH prior to
chromatography: the resulting alcohols (5a-, 7a- and 7b-OH-
Carotenoids were extracted from irradiated lipoprotein sam-
ples loaded with either Zn- or Pd-BPheid, and investigated by
HPLC analyses for oxidation products. The three most
abundant carotenoids in both LDL and HDL were b-crypto-
xanthin (t
(t
r
r r
: 21.2 min), lycopene (t : 23.0 min) and b-carotene
: 24.0 min). No additional carotenoids could be detected
after irradiation in untreated lipoproteins (data not shown).
In LDL (Fig. 4a) and HDL (see supplementary material,
Fig. S5a) loaded with Zn-BPheid, one additional carotenoid
band was detected. The product (t : 22.7 min) had a typical
r
three-banded carotenoid absorption (kmax = 409 [shoulder],
4
30 and 454 nm, Fig. 4b). The blueshift of all bands is
indicative of a shortened conjugation system as is present, for
example, in 7,8-dihydro-b-b-carotene (kmax = 410 [sh], 429
and 454 nm) (65). Two oxidation products of b-carotene have
a similar absorption spectrum: 5,8-epoxy-5,8-dihydro-b-b-
carotene (mutatochrome) and 5,8-endoperoxy-5,8-dihydro-b-
b-carotene (5,8-endoperoxy-b-carotene, Fig. 5); the latter was
identified by several groups as a main reaction product of
1
b-carotene with O (33,66–68).
2
The concentration of this product increased seven-fold
if lipoprotein samples were irradiated in the presence of
Pd-BPheid instead of Zn-BPheid: three additional oxidation
products were found in low concentrations (Fig. 4d) and all
had carotenoid-like absorption spectra (Figs. 4c and S5b). The
pigment eluting at 19.0 min had an absorption spectrum
(
kmax = 409 [sh], 430 and 455 nm) that is almost identical to
that of the 5,8-endoperoxide; judged from its mobility,
however, it is possibly a more polar derivative with further
modifications outside the conjugation system. The pigment
eluting at 21.0 min had an even shorter conjugation system
(kmax = 381, 402 and 428 nm), resembling that of 7,8,7¢,8¢-
tetrahydro-w,w-carotene (f-carotene; kmax = 378, 401 and
Figure 3. HPTLC of lipids extracted from irradiated, pigment-loaded
lipoprotein fractions after treatment with CholEase (a, TMPD
staining) and after treatment with CholEase and NaBH (b, CuSO
4 4
staining). Lipoprotein fractions were obtained by SDGU fractionation
of BP incubated with the respective pigment and irradiated with NIR
)
2 )1
light (low-pass filter RG695; 530 lE m
BPheid; 2: LDL + Zn-BPheid; 3: HDL + Pd-BPheid; 4: HDL +
s ; 60 min). 1: LDL + Pd-
4
26 nm) (46) or 5,8,5¢,8¢-diendoperoxy-b-carotene; the latter
Zn-BPheid; S1: 5a-OOH-Chol standard; S2: 5a-OH-Chol standard; S3:
was identified as a photo-oxidation product of b-carotene with
O2 in solution (33). The third minor product had an
7-OH-Chol standard. Top bands (esters) in lanes 1–4 could contain
1
traces of (5a-)OH esters in (b).

3
46 J o¨ rg Dandler et al.
Figure 4. Photosensitized products of endogenous carotenoids of blood plasma. HPLC of extracts of LDL loaded with (a) Zn-BPheid (12 lM) or
c) Pd-BPheid (13 lM) before and after irradiation with NIR light (low-pass filter RG695; 530 lE m)
s ) for 10 min (a) or 180 min (c), and in situ
2
)1
(
absorption spectra (b, d) of the respective main oxidation products (retention times are marked with arrows in the HPLC chromatograms). BP was
pigment-loaded prior to LDL preparation by SDGU. Chromatograms of the irradiated samples were shifted up by 0.4 units. See Fig. S5 for the
respective data obtained in HDL.
absorption spectrum (kmax = 453 [sh], 473 and 504 nm)
indicative of a longer conjugation system that is present, for
example, in lycopene (kmax = 449 [sh], 473 and 504 nm), but
the product is more polar than the latter judged from the
retention time of 20.6 min. It is possibly a lycopene oxidation
product in which the p-electron system has not been shortened,
but which is instead modified at an end group without
affecting the p-system.
2,3-dichloro-5,6-dicyano-p-benzoquinone (69). The reaction is
analogous to the oxidation of BChl a to [3-acetyl]-chlorophyll
a (70); dehydrogenation of ring B is favored over dehydroge-
nation of ring D for steric reasons (69,71,72). Based on the
absorption changes and the known extinction coefficients, less
than 50% of Pd-BPheid was photo-oxidized to [3-acetyl]-Pd-
Pheid, while the remainder formed products with little
absorption in the VIS ⁄ NIR range. This is similar to results
obtained with BChl-derivatives in organic solvents (12). There
was, in particular, no spectroscopic or HPLC indication of a
further photo-oxidation of the chlorin to the respective
porphyrin derivative. This is not only due to the aforemen-
tioned preferential reaction at ring B but also to the NIR
irradiation conditions (695 nm low-pass filter) that provide
only little light in the absorption range of [3-acetyl]-Pd-Pheid
(kmax = 660 nm). There was also no indication of the forma-
tion of cation radicals, which have a broad, low-intensity NIR
absorption band (73).
Chlorophyllous oxidation products
When irradiating LDL (Fig. 6a) or HDL fractions (data not
shown) loaded with Pd-BPheid (kmax = 760 nm), the corre-
sponding chlorin derivative, [3-acetyl]-Pd-pheophorbide ([3-
acetyl]-Pd-Pheid; kmax = 660 nm), was the only detectable
oxidation product with significant absorption in the VIS ⁄ NIR
range. It was identified by co-chromatography with an
authentic sample obtained from Pd-BPheid by oxidation with
Further oxidation to the porphyrin, however, is also
dependent on the environment: it did occur if Pd-BPheid was
irradiated under otherwise identical conditions in CES, a
mixture of CrEL, ethanol and (normal) saline that is used as a
solubilizing system for tetrapyrroles into BP. The oxidation
product,
[3-acetyl]-Pd-protopheophorbide
([3-acetyl-Pd-
PPheid; kmax ꢀ600 nm) (Fig. 6b), was previously reported as
a photoproduct of Pd-BPheid in 3% Triton X-100 ⁄ chelexed
PBS (42).
Deuterium isotope effect
WST09 is very stable in NIR light. When an HDL fraction
Figure 5. Structures of b-carotene (top) and 5,8-endoperoxy-5,8-di-
hydro-b-carotene (bottom).
2
loaded with WST09 was dialyzed against H O and irradiated

Photochemistry and Photobiology, 2010, 86 347
Figure 6. Photoreaction of WST09 in a dialyzed LDL fraction (a) and
in CES (b). Absorption spectra before and during irradiation with NIR
)
2 )1
light (low-pass filter RG695; 530 lE m
s ).
Figure 7. Photobleaching kinetics of WST09 (a) and WST11 (b) in
2
lipoproteins (HDL). Pigment-loaded HDL was dialyzed against H O
)
2 )1
or D
different time scales in (a) and (b).
2
O, and irradiated with NIR light (530 lE m
s ). Note the
in an open cuvette with the maximum light intensity of our
)
2 )1
apparatus (NIR light intensity: 530 lE m
s ), 50%
bleaching was observed after 25.5 min (Fig. 7a). The high
stability of this Pd-complex is similar to that reported for
Pd-bacteriopheophytin (Pd-BPhe) in organic solvents (11,12).
WST11 is, by contrast, much more photolabile: at the same
light intensity it was 50% bleached within only 2.1 min
dialyzed in exactly the same way against H O, which ensured
2
that the concentrations of natural, water-soluble antioxidants
were reduced to a similar extent in both samples.
Some lipophilic antioxidants will remain in the HDL after
dialysis. To test if the unusual isotope effect is inherent in the
HDL fraction, WST09 was irradiated in micelles of CrEL.
This polyethylene glycol derivative of castor oil is frequently
used in medicine as a substitute for DMSO, although it is not a
completely inert substance (74). WST09 was first dissolved at
very high concentration (ꢀ720 lM) in a mixture of CrEL and
ethanol (1:1 vol ⁄ vol), and this solution was subsequently
diluted (1:39 vol ⁄ vol) with either H O or D O. In this artificial
(
Fig. 7b).
When the same experiments with WST09 and WST11 were
performed in D
rapid bleaching than in H O, but this effect was reversed after
2
O, there was, in both cases, initially a more
2
longer periods when the bleaching was inhibited in D
compared with H O. This reversal effect is more pronounced
with the less polar WST09: at the onset (<3 min) the
bleaching in was much faster than in
⁄ k = 2.25; all given k ⁄ k values are initial rate con-
stants extrapolated to t = 0), but was then almost arrested in
O at a level of ꢀ83% of the original pigment content
Fig. 7a). This reduced kinetics is not due to the depletion of
oxygen, because in H O the pigment is fully bleached after
approximately 1 h. The effect is qualitatively similar with
WST11 (k ⁄ k = 1.75), but less pronounced (Fig. 7b). Care
2
O
2
D
2
O
2
H O
2
2
(
k
D
H
D
H
system that contains unsaturated fatty acids but is otherwise
devoid of antioxidants, the same two phases of the illumina-
tion kinetics were observed (Fig. 8), albeit less pronounced
than in HDL. The initial bleaching (<3 min) was still faster in
D O than in H O (k ⁄ k = 1.95), and it leveled off in D O at
D
2
(
2
2
2
D
H
2
ꢀ52% of the original pigment content.
D
H
Both the lipoprotein and the CrEL systems are heteroge-
neous; it was, therefore, of interest to study the isotope effect
on photobleaching also in a homogeneous system, such as in
water. The high solubility of WST11 allowed such studies,
was taken that the samples were comparable, except for the
replacement of H O and D O. For isotope exchange, HDL
2
2
2 2
samples were dialyzed against D O. The H O-controls were

3
48 J o¨ rg Dandler et al.
not require any additions to the samples, and its photoprod-
ucts have been established (32).
Identification of 5a-OOH-Chol after irradiation of both
LDL and HDL gave strong confirmatory support for a Type II
mechanism using Pd-BPheid or Zn-BPheid as photosensitizer.
The HPTLC analysis of the reaction mixtures was, nonethe-
less, unsatisfactory: the separation of the peroxides is poor and
the products are relatively labile. Free Chol is furthermore
accompanied by Chol-esters, and these as well as their photo-
oxidation products are not included in the standard analysis.
We therefore modified the assay in two ways. First, the esters
were hydrolyzed (nearly) quantitatively to the respective free
Chol derivatives by treatment with CholEase, and second the
product peroxides were reduced with borohydride to the
respective alcohols, 5a-OH-Chol and the two epimers of 7-OH-
Chol. The HPTLC separation of these products was much
clearer, and the products can be identified more rigorously by
co-chromatography with stable authentic samples that are
accessible by established procedures (49,50). Of particular
importance was the resolution of the 7-OH-Chol epimers. The
Figure 8. Photobleaching kinetics of WST09 in a micellar environ-
ment. WST09 was dissolved in a mixture of CrEL and ethanol (1:1
vol ⁄ vol), subsequently diluted with H O or D O (1:39 vol ⁄ vol) and
2 2
)1
)
2
irradiated with NIR light (530 lE m
s ).
5
a-OH-Chol standard contained small amounts of 7a-OH-
Chol, while 7b-OH-Chol is absent. It is also known that 5a-
OOH-Chol can be transformed into 7a-OOH-Chol via an
allylic rearrangement, while the 7b-epimer is formed much
more slowly by subsequent epimerization to 7b-OOH-Chol
(
75,76). 7-OOH-Chol generated by a Type I reaction is, by
contrast, an epimer mixture that yields, after reduction, the
two epimeric alcohols, as does reduction of 7-keto-Chol
(49,50). The consistent lack of spots at the position of 7b-
OH-Chol is, therefore, the unequivocal evidence for a Type II
reaction of both sensitizers.
1
In principle, O
2
can also be generated by disproportion-
ation of peroxyl radicals (32,77) but, in this case, formation of
some 7-OOH-Chol is expected by a competing direct action of
the latter. The consistent absence of 7b-OH-Chol, the reduc-
tion product of 7b-OOH-Chol, also argues, therefore, against
such an indirect production of O . Thus, we conclude that
1
2
only 5a-OOH-Chol is formed in the lipoprotein samples upon
irradiation and that Chol in lipoproteins is therefore attacked
by Type II reactions, irrespective of the use of Pd- or Zn-
BPheid as photosensitizer.
2 2
Figure 9. Photobleaching kinetics of WST11 in H O and D O.
)
2 )1
Solutions were irradiated with NIR light (12 lE m
s ).
While these results agree with the action of many photo-
sensitizers, a more complicated mechanism has been suggested
by Vakrat-Haglili et al. (42). Using sodium azide as quencher,
the authors concluded that Pd-BPheid is oxidized to the
while WST09 is only poorly soluble in water. Photobleaching
of WST11 gave a positive deuterium isotope effect throughout
the reaction (k
completely bleached in both D O and H O (Fig. 9). Thus,
D
H
⁄ k = 1.50), and the pigment was eventually
chlorin level by superoxide or peroxide radicals, and only the
. It
2
2
1
subsequent oxidation to the porphyrin level involves O
2
these results indicate that the unusual biphasic deuterium
isotope effect is related to the heterogeneous amphiphile-in-
water reaction medium.
should be noted, however, that photobleaching of the sensi-
tizer, or its chlorin oxidation product, are only minor reactions
in plasma lipoproteins that are rich in oxidizable components
that compete for the oxidant. In a recent study, it has been
estimated that the concentration of peroxides at the end of the
irradiation (160 lM) is ca 18-fold higher than the initial
concentration of the sensitizer (9 lM) (43). The chlorin,
DISCUSSION
Photodynamic action of bacteriopheophorbides in lipoproteins
is dominated by the Type II mechanism
[3-acetyl]-Pd-Pheid which, under our conditions, amounts to
Investigation of the mechanism, by addition of quenchers,
indicated a Type II reaction mechanism and this was sup-
ported by a more rigorous test relying on the formation of
mechanism-dependent reaction products of a probe substrate.
A particularly convenient substrate, in the present case, was
cholesterol: as a natural component of lipoproteins (55) it does
about half the concentration of Pd-BPheid originally present
therefore constitutes only about 3% of the products.
Such a model also explains the much higher stability of
(monomeric) BChl derivatives in lipoproteins, compared to the
situation in detergent micelles or in solution where such
competitors are missing (43). In pure detergent environments

Photochemistry and Photobiology, 2010, 86 349
(
CES; Triton X-100) that are poor as competing substrates, the
possible, but ultimately unsatisfactory, explanations: (1) First,
bacteriochlorin sensitizers are much more rapidly oxidized to
chlorins (and other products), and the chlorins are further
photo-oxidized to porphyrins. The relevance of competing
target molecules, such as Chol(esters) (see above), unsaturated
fatty acids and reactive amino acids, is further supported by
the observation that the oxygen consumption upon irradiation
increases with increasing lipoprotein concentrations (data not
shown).
2 2
that oxygen is more rapidly depleted in D O than in H O
because the larger lifetime means a larger range of actions,
thus allowing access to more target molecules like cholesterol
(esters), unsaturated fatty acids, oxidizable amino acids and
lipophilic antioxidants. Such target molecules are absent,
however, or at least much less abundant in the CrEL-system,
while the same biphasic behavior is observed. (2) Second, that
the duration of residence of oxygen near the sensitizer is
In summarizing these results, we suggest that lipoproteins
have a considerable capacity for quenching Type II photore-
actions, and that at least the peroxides of Chol and its esters
decreased in D O to such an extent that it compensates the
2
1
increased lifetime of O
2
. This seems unlikely even if it involves
H-bonding. While this cannot be excluded, it would still not
explain the mechanistic switch. (3) Third, the change from
1
1
are exclusively formed by reaction with O . In cells, O is
2
2
mainly quenched by proteins (78) but this may be true only for
quite hydrophilic photosensitizers and environments. For
more hydrophobic photosensitizers including Pd-BPheid that
are embedded in membranes, a higher participation of lipids
and other nonpolar substances can be expected. Last but not
least, the resulting photo-oxidation products may be still quite
reactive and may require further attention regarding their
participation in photodynamic action. Such a complication has
previously also been suggested for carotenoids: b-carotene can
bind up to eight oxygen atoms (33). Absorption spectra of the
major and of two of the three minor photoproducts match
with those of endoperoxides reported in the latter work.
2 2
H O to D O may induce a phase transition, or accelerate one
that is induced by ROS. However, in membranes containing
unsaturated fatty acids and cholesterol no isotope effect on the
molecular organization of the lipid membranes could be
detected (81), and it is unlikely that a similar effect would
operate in lipoproteins and in the CrEL system. (4) Fourth, it
is conceivable that the dehydrogenation reaction is affected. As
only a fraction of the pigments is oxidized to chlorins, an
isotope effect of the observed size also seems unlikely. (5)
Finally, bleaching during the late phase, where oxygen
concentration is low, may relate to a mechanism that is
1
independent of
mechanism (82). Although isotope effects have been reported
2
O , but rather relies on a Type I or other
not only for the lifetime but also for the yield of triplets (and
1
O ) (83–85), there is, to our knowledge, no report on a
2
A mechanistic switch at low oxygen concentration
Notwithstanding the evidence for Type II as the main
photosensitizing mechanism in lipoproteins, the H ⁄ D isotope
experiments point to a mechanistic switch at low oxygen
negative D-isotope effect on these processes.
Because none of the above five hypotheses can satisfactorily
explain the negative deuterium isotope effect, we propose,
therefore, an alternative explanation that involves two factors:
first, the second phase of bleaching is not due to the action of
concentrations. For many porphyrin sensitizers, the isotope
lifetime is not an important limiting factor for
⁄ k = 1.2–1.6) (79,80). These data indi-
1
effect on the O
2
1
1
photobleaching (k
D
H
2 2
O , and second, the equilibrium concentration of O during
cate that the unusual biphasic deuterium isotope effect is
related to the heterogeneous amphiphile-in-water reaction
medium; in agreement with this is its absence in water.
The unusual isotope effect is more pronounced with WST09
than with the more hydrophilic WST11; that is, with WST09
there is a much smaller amplitude than with WST11 of the
irradiation is so high with respect to the total oxygen
3
concentration that the amount of triplet oxygen ( O ) is
2
significantly reduced. A mechanistic switch has also been
previously reported for hematoporphyrin: at low concentra-
tions (ꢀ1 lM) Type II reactions prevail, while at higher
pigment concentrations (ꢀ15 lM) Type I photoreactions
dominate. Aggregation has been invoked for this mechanistic
switch (86), which does not, however, apply to all the BChl
derivatives studied in this work (41). Alternatively, Type I
photoreactions are favored at low pigment ⁄ oxygen ratios,
because of the higher probability for primary reactions with
molecules other than oxygen (82).
phase with the normal, positive D-isotope effect that is
1
expected for a O -induced bleaching (and indeed observed
2
in water). Therefore, this part of the photobleaching process
seems to be related to a fraction of the pigment that resides in a
largely aqueous environment near the surface of the HDL
particles, or even free in solution. The presence of substantial
amounts of free pigment is unlikely, because there was no loss
of pigment during dialysis. We therefore conclude that the
pigment fraction showing the positive isotope effect is located
near, or exposed to the aqueous phase. The second pigment
fraction exhibiting the negative D-isotope effect of photo-
bleaching would then be located in the more hydrophobic
interior. This is more readily accessible for WST09 than for the
more polar WST11: while >80% of WST09 is located in the
lipoprotein fractions (LDL and HDL) of human BP, ꢀ80% of
WST11 is bound to HDP, most probably human serum
albumin (41).
3
The second precondition is that the reduction of O in D O
2
2
relative to that in H O at low oxygen concentrations can be
2
3
met when O is more rapidly excited to O than when the
1
2
2
3
latter relaxes back to O . The extended lifetime of O in D O
1
2
2
2
3
would, in this case, reduce the amount of O present further
2
than is the situation in H O, thus generating a negative isotope
2
effect for reactions depending on the latter. At a quantum flux
)1
of 530 lE m)
2
s , and a pigment concentration of 18 lM,
1
)
each pigment molecule is excited ꢀ3 times s . With a
quantum yield of 99%, this could generate ꢀ3 molecules of
1
)1
O s . At saturating O conditions (250 lM), this excitation
2
2
While this model can rationalize the extent and properties of
the first phase of bleaching with a positive D-isotope effect, we
are currently unable to conclusively explain the negative
isotope effect in the later phase. We first considered several
rate would be far too low because O2 is present at an
approximately 14-fold molar excess over the sensitizer; how-
ever, O2 is depleted in the case of WST09 in HDL within
ꢀ2.5 min (measured by a Clark-type electrode; data not

3
50 J o¨ rg Dandler et al.
shown) and can only be restored by diffusion, which is further
inhibited in the heterogeneous lipid system. A reduction to
values of 1% and less is also compatible with in vivo reports
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. HPTLC of lipids extracted from irradiated,
pigment-loaded lipoprotein samples before incubation with
CholEase (TMPD staining).
using a bacteriopheophorbide-serine as sensitizer (87). Assum-
O, a reduction in the oxygen
1
ing an O
2
lifetime of 60 ls in D
2
4
concentration by a factor of 10 would suffice to maintain a
substantial fraction of the total oxygen in the singlet state. This
model, therefore, seems to be compatible with the experimental
results.
Figure S2. HPTLC of lipids extracted from lipoprotein
samples after incubation for 24 h with an acidified solution of
CuSO (TMPD staining).
4
Figure S3. Solvent dependence of cholesterol hydroperox-
ides generated by irradiating solutions of WST09 (Pd-BPheid,
CONCLUDING REMARKS
1
hydroperoxides.
Figure S4. Solvent dependence of cholesterol hydroperox-
ides generated by irradiating solutions of WST09 (Pd-BPheid,
11 lM) and cholesterol (13 mM): Analysis of cholesterol
hydroxides.
Figure S5. Photosensitized products of endogenous carote-
noids of blood plasma.
Table S1. Effect of ROS quenchers on the photostability of
1 lM) and cholesterol (13 mM): Analysis of cholesterol
A Type II mechanism was previously established for photo-
sensitization by transmetalated BChl derivatives in solution
(11). This has now been extended to the two lipoprotein
fractions, LDL and HDL, that carry the majority of these
pigments in the bloodstream (41). The only exception to this
rule is the water-soluble taurinated derivative, WST11, for
which a Type I mechanism has been proposed (42,88). This
difference in mechanism may be a direct result of the chemical
modification, but can also be due, indirectly, to the hydrophilic
microenvironment preferred by the latter pigment that is also
responsible for its enrichment in the HDP fraction of BP (89).
)1
Zn-BPheid (10 lM) in HDL (8 g L ) and Pd-BPheid (6 lM) in
LDL (8 g L) ).
1
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
The mechanistic switch seen for WST09 and WST11 at low O
concentrations is compatible with such a dominant microen-
2
vironmental effect. Such a switch at low oxygen concentra-
to one involving
1
tions, from a Type II reaction involving O
2
other ROS, may be of practical significance for PDT and thus
warrants further investigation.
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