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www.elsevier.com/locate/jphotobiol
Journal of Photochemistry and Photobiology B: Biology 75 (2004) 157–163
A method for separating ALA from ALA derivatives usingionic exchange extraction
Gabriela Di Venosa, Haydee Fukuda, Christian Perotti, Alcira Batlle 1, Adriana Casas *
Centro de Investigaciones sobre, Porfirinas y Porfirias (CIPYP), CONICET and FCEN, University of Buenos Aires, Ciudad Universitaria,
Pabellon II, 2do piso; (1428) Capital Federal, Argentina
Received 28 November 2003; received in revised form 13 May 2004; accepted 5 July 2004
Available online 7 August 2004
Abstract
Photodynamic therapy using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX is a recent approach to detect and treat
some malignancies. The use of lipophilic derivatives of ALA has been exploited in the last years to enhance ALA penetration. In this
paper, we describe the application of the Mauzerall and Granick�s method [J. Biol. Chem. 219 (1956) 435] to the quantification of
ALA derivatives. We also describe the employment of reusable ion-exchange chromatographic columns for separating mixtures of
ALA and ALA derivatives present in biological samples. The relation between 555 nm absorbance and ALA or ALA derivative
concentration was linear up to 100 nmol/ml and the limit of detection of ALA and ALA derivatives was 1 nmol per ml. We em-
ployed a Dowex 50 X8 hydrogen form resin to separate ALA from the derivatives. Whereas 90 ± 4% of the total ALA was eluted
using sodium acetate, only 3–9% of the ALA derivatives was recovered. Only upon exposure of the resin to a high HCl concentra-
tion, the ALA derivatives were completely released. We employed this new method for the separation of ALA from ALA derivatives
in cells exposed to different ALA compounds.
� 2004 Elsevier B.V All rights reserved.
Keywords: Photodynamic therapy; PDT; Aminolevulinic acid; ALA; ALA derivatives; ALA esters; Dowex resin
1. Introduction
Photodynamic therapy (PDT) using 5-aminolevulinic
acid (ALA)-induced protoporphyrin IX is a recent ap-
proach to treat neoplasms in a variety of organs [2–9].
Besides, ALA-induced porphyrin fluorescence may also
assist in the early detection of some malignancies [10].In the cytosol two molecules of ALA are combined to
form porphobilinogen (PBG) and four molecules of
PBG are then bound to form uroporphyrinogen. Next,
uroporphyrinogen is converted to coproporphyrinogen,
1011-1344/$ - see front matter � 2004 Elsevier B.V All rights reserved.
doi:10.1016/j.jphotobiol.2004.07.001
* Corresponding author. Tel./fax: +54-11-4782-7963.
E-mail addresses: [email protected] (A. Batlle),
[email protected] (A. Casas).1 Present address: Viamonte 1881 10A, 1056 Buenos Aires, Argen-
tina. Fax: +54-11-4811-7447.
which is taken up by mitochondria to form protoporph-
yrin IX, which is the photosensitizing molecule.
The hydrophilic nature of the ALA molecule limits
somehow the penetration through the skin stratum cor-
neum. Hence, the use of more lipophilic derivatives of
ALA which were expected to have better penetrating
properties has been exploited in the last five years [11].Mauzerall and Granick in 1956 [1] developed a simple
colorimetric method for the quantitative determination
of ALA and PBG in urine, and this procedure is com-
monly used still nowadays, in the diagnosis of porphy-
rias. For ALA quantification the procedure was based
on the condensation of the compound in presence of
acetylacetone to give a pyrrole which can react with
the Ehrlich reagent. The Ehrilch reagent, also knownas Ehrlich diazo reagent contains dimethylaminobenzal-
dehyde (DMAB) in acid solution. In the usual
N OH
H
O
O
Undecanoyl-ALA
O
O
N O(CH2)5CH3
H
O
O
O
N O
H
R,S -ALA-2 (hydroxymethyl)tetrahydropyranyl ester
N OCH3
H
Methyl-ALA
Hexyl-ALA
O
O
N O(CH2)10CH3
H
O
O
ALA
Fig. 1. Structures of ALA and the ALA derivatives.
158 G. Di Venosa et al. / Journal of Photochemistry and Photobiology B: Biology 75 (2004) 157–163
colorimetric procedure of the Ehrlich reaction, the
amine of the pyrrole reacts with the aldehyde of DMAB
in acid solution to form a colored condensed product.
The increasing interest on the use of ALA derivatives
in PDT and the elucidation of the mechanisms of action
involved led us to develop a method for separating ALAfrom ALA derivatives in biological samples.
We found [12] that in cell lines exposed to saturating
concentrations of ALA hexyl ester, the amount of ALA
and/or ALA hexyl ester was 4 times higher than that
accumulated from ALA. However, neither PBG nor
porphyrin synthesis was higher. Since the aim of the
use of ALA derivatives is to improve protoporphyrin
IX synthesis, it is crucial to elucidate the limiting stepon the conversion of ALA esters to porphyrins. In order
to investigate if the esterases were limiting ALA deriva-
tives conversion to porphyrins, we developed a method
for separating ALA from ALA derivatives in cell lines.
In this paper, we describe the application of the
Mauzerall and Granick�s method to the quantification
of ALA derivatives. Besides, we also describe the
employment of reusable ion-exchange chromatographiccolumns for the separation of mixtures of ALA and
ALA derivatives present in biological samples.
2. Materials and methods
2.1. Chemicals
ALA, ALA-methyl ester (Me-ALA), Dowex 50 X8
resin, 100–200 mesh, and Dowex 50W x 2-200(H)
hydrogen ionic form were obtained from Sigma Chem
Co.ALA derivatives were obtained as the hydrochloric
salts. ALA hexyl ester (He-ALA) and Undecanoyl-
ALA (Fig. 1) were synthesized according to the method
of Takeya [13] by reacting ALA with hexanol and unde-
canol, respectively, in the presence of thionyl chloride.
The mixture was stirred at 70 �C until ALA.HCl was
completely dissolved and the reaction was confirmed
by TLC (Cl2CH2/MeOH 9:1). The excess alcohol wasevaporated under high vacuum. After addition of dieth-
ylether, the HCl salts of the ALA esters were allowed to
crystallize at 4 �C. Yields ranged from 40% to 60%.
R, S-ALA-2-(hydroximethyl)tetrahydropyranyl ester
(THP-ALA) was similarly prepared. The crude product
was purified by flash column chromatography on silica
gel eluting with Cl2CH3/MeOH mixtures. The yield
was about 20%.Purities of the synthesized compounds were always
higher than 95%, as established by thin layer chroma-
tography (TLC) and NMR techniques.
ALA derivatives were dissolved in water immediately
before using.
2.2. Validation of ALA derivatives determination
Different amounts of ALA or ALA derivatives
freshly dissolved in water were added to 1 M acetic ace-
tate buffer, pH 4.8. Then they were condensed with ace-tylacetone in a boiling water bath during 10 min. Once
cooled, an aliquot was mixed with an equal volume of
the regular Ehrlich reagent. The absorbance was read
at 555 nm (between 8 and 15 min after mixing), in a
Hewlett Packard diode array Spectrophotometer model
8452A. The Ehrlich reaction products have a maximal
absorption intensity at 555 nm for all the ALA
derivatives.
2.3. Characterization of He-ALA products after conden-
sation and Ehrlich reactions
To discard the hypothesis of He-ALA hydrolization
to ALA during the condensation reaction, we condensed
He-ALA with acetyl acetone and the reaction product
was lyophilized overnight so that acetyl acetone excesswas completely removed. The dryed compound was re-
acted with hydoxylamine giving a red colored com-
pound, which reveals the presence of an ester group
according the identification reaction described in Shri-
ner et al. [14]. In a parallel set of experiments, condensed
ALA gave no colored reaction.
In additon, we examined possible He-ALA hydroliza-
tion by Ehrlich reagent itself or by heat action alone. Weboiled a He-ALA solution in acetic acetate buffer for 10
min and then added an equal volume of Ehrlich reagent.
G. Di Venosa et al. / Journal of Photochemistry and Photobiology B: Biology 75 (2004) 157–163 159
The resulting compound was dialyzed overnight to elim-
inate salts, filtrated to eliminate DMAB and lyophilized
afterwards. The presence of He-ALA was confirmed by
TLC employing acetone:methanol (6:4) and was re-
vealed with ninhydrin.
2.4. Separation of ALA from ALA derivatives by cationic
interchange chromatography
Three grams of Dowex resin was washed several
times and treated with 5 ml of 4 N HCl, then 5 ml of
1 N HCl and 5 ml of water and placed into plastic col-
umns (Bio-Rad). A 3 ml aliquot of the ALA or ALA
derivative solution prepared in 5% TCA was appliedto the top of the columns and allowed to drain through.
Then 7 ml of 1 M sodium acetate was added ml by ml
and the eluates were collected separately. Then 0.25 ml
of each eluate was mixed with 0.25 ml of 1 M acetic ace-
tate buffer, pH 4.8, and 0.05 ml of acetyl acetone. Con-
densation and quantification was performed as
described previously.
Under these elution conditions the ALA derivativeswere retained in the column; then they were eluted with
10 ml of 10 M HCl added ml by ml. Then 0.1 ml of each
ml of eluate was mixed with an equal volume of 10 M
NaOH to adjust the pH, except for the eluate 1, which
did not need NaOH addition. Then 1 M acetic acetate
buffer, pH 4.8, was added to complete 0.5 ml and finally
acetyl acetone was added for condensation as described
above.The resin was prepared for its reuse by adding succes-
sively 5 ml of 4 N HCl, 5 ml of 1 N HCl and 5 ml of
water.
2.5. Cell line and cell culture
Cell line LM3 [15] derived from the murine mammary
adenocarcinoma M3 was cultured in Minimum essentialEagle�s medium, supplemented with 2 mM LL-glutamine,
40 lg gentamycin/ml and 5% fetal bovine serum (FBS),
and incubated at 37 �C in an atmosphere containing 5%
CO2. Cells were used 48 h after plating.
2.6. ALA, ALA derivatives and PBG determinations in
cells
LM3 cells were seeded in 100 mm dishes. After 72 h,
medium was removed and cells were exposed for 3 h to
0.6 mM ALA or ALA derivatives in medium without
serum. Afterwards, cells were washed 4 times with
PBS and 5% TCA was added. After scrapping, the cells
were centrifuged and the supernatant was employed for
ALA and PBG determinations. Membrane disruption
with Triton X-100 or sonication previous to TCA treat-ment proved not to increase the release of ALA or ALA
derivatives from cells.
For PBG determination, the Ehrlich�s reagent was
added to the deproteinized TCA supernatant. ALA val-
ues were obtained by subtracting PBG values from the
total of condensed pyrroles.
In parallel, a pool of supernatant from two dishes (3
ml) was passed through a column of Dowex 50 X8 resin.Five ml of water was added in order to elute PBG. ALA
was eluted with 7 ml of 1 M sodium acetate and ALA
derivatives with 10 ml of 10 N HCl as explained above.
The percentage of free ALA was calculated from the to-
tal ALA + ALA esters after subtracting the PBG contri-
bution.
2.7. TLC of ALA and ALA derivatives:
The sodium acetate and HCl eluates from ALA deriv-
atives were dialyzed overnight to eliminate salts and
lyophilized afterwards. The presence of ALA and
ALA derivatives was confirmed by TLC. TLC was per-
formed using acetone:methanol (6:4) and was revealed
with ninhydrin.
2.8. Statistical analysis
Each experiment was performed in triplicates. Data
are presented as means ± SD.
3. Results
3.1. Validation of ALA derivatives determination
The relation between 555 nm absorbance and ALA
or ALA derivatives concentration was linear up to 100
nmol/ml (Fig. 2). The standard deviation from the mean
was identical for both ALA and its derivatives (less than
5%). The limit of detection of ALA and ALA derivatives
was 1 nmol per ml.The absorbance spectra of the Ehrlich reaction prod-
ucts were identical for both ALA and ALA derivatives,
showing that the Ehlich reaction indistinguishably de-
tects ALA and ALA derivatives. However, in order to
gain insight into the nature of ALA derivatives products
of condensation and Ehrlich reactions we performed
two tests employing He-ALA. First, we condensed He-
ALA with acetly acetone and afterwards, we were ableto identify the ester group, showing that the condensa-
tion did not hydrolized the ALA derivative. In addition,
we exposed He-ALA to Ehrlich reagent and to 10 min
heating and we identified by TLC the presence of He-
ALA and not ALA, discarding the hypothesis of hydro-
lization of the ester, at least in a high extent, in highly
acidic conditions and heat.
ALA and ALA derivatives were determined asdescribed in Section 2. Nanomoles of ALA or ALA
0 10 20 30 40 50 60 70 80 90 100
0.25
0.50
0.75
1.00
1.25
1.50
1.75
ALAHe-ALAUndecanoyl-ALATHP-ALAMe-ALA
nmoles ALA or ALA derivative/ml
Abs
555
nm
0 1 2 3 4
0.02
0.04
0.06
0.08
0.1
0
Fig. 2. Validation of the method for ALA derivatives.
160 G. Di Venosa et al. / Journal of Photochemistry and Photobiology B: Biology 75 (2004) 157–163
derivatives are expressed per ml of reaction mixture be-
fore adding the Ehrlich�s reagent.
3.2. Elution of ALA and ALA derivatives by cationic
interchange chromatography
Table 1 shows the percentage of recovery of ALA and
ALA derivatives by ionic interchange chromatography
after sodium acetate elution. Three solutions of ALA
or ALA derivatives were passed through the columns.
The recovery of ALA in the sodium acetate eluate
was around 90 ± 4% for the three concentrations tested,
and 85% of the amount recovered was eluted in the 3–6
ml fractions. Considering the limit of detection of theMauzerall and Granick method which is, under our con-
ditions, 1 nmol/ml and taking into account the buffer
dilution, a minimum of 2 nmol/ml eluate is detected.
According to these calculations, the limit of detection
of ALA by this method is 9.5 nmol, as long as no less
than 2 nmol are recovered per each of the 3, 4, 5 and
Table 1
Percentage of recovery of ALA or ALA derivatives in the sodium
acetate eluates
6 lmol 0.6 lmol 0.06 lmol
ALA 89.1 ± 4.3 91.2 ± 3.2 94.4 ± 5.2
He-ALA 5.4 ± 0.36 6.4 ± 0.35 2.7 ± 0.18
Me-ALA 28.3 ± 0.15 27.5 ± 0.12 21.81 ± 0.11
Undecanoyl-ALA 6.8 ± 0.25 5.3 ± 0.14 6.4 ± 0.21
THP-ALA 9.2 ± 0.45 11.3 ± 0.53 2.5 ± 0.07
Different amounts of ALA or ALA derivatives (6, 0.6 and 0.06 lmol)
were passed through a Dowex 50 X8 resin and eluted with 7 ml of 1 M
sodium acetate. Each ml of eluate was quantified for ALA/ALA
derivative detection according to Section 2. The percentage of ALA or
ALA derivatives recovered was calculated.
6 fraction of eluate. However, due to the intrinsic varia-
tion of the ALA concentration in the eluates, slightly
less than 2 nmol may be released, thus raising the limit
of detection to 12 nmol of ALA instead of 9.5.
Only 3–9% of three of the ALA derivatives was
recovered in the sodium acetate fractions at all the con-centrations tested. This low percentage of the total very
likely corresponds to free ALA, resulting as a product of
either the hydrolization of the ALA esters in the acidic
conditions of the column or to a small percentage of free
ALA present as an impurity in the ALA esters. Instead,
20–30% of the methyl ALA ester was recovered in the
sodium acetate fractions.
In this set of experiments TCA was used to dissolveALA or ALA derivatives, as this acid was required to
precipitate the cell proteins; identical results were ob-
tained when the ALA or ALA esters were dissolved in
water.
In order to release the ALA derivatives bound to the
column, solutions of higher ionic strength were used.
Table 2
Percentage of recovery of ALA or ALA derivatives in the 10 M HCl
eluates
6 lmol 0.6 lmol 0.06 lmol
ALA 15 ± 0.77 0 0
He-ALA 95.6 ± 3.82 102 ± 6.12 83.4 ± 4.97
Me-ALA 71.5 ± 2.15 68.7 ± 4.33 73.6 ± 3.18
Undecanoyl-ALA 98.3 ± 5.30 95.4 ± 5.77 75.6 ± 4.64
THP-ALA 97.6 ± 4.15 101 ± 6.28 77.8 ± 3.87
After the sodium acetate treatment of Dowex columns treated as ex-
plained in previous figure, 10 ml of 10 M HCl was added for elution of
ALA derivatives. Each ml of eluate was quantified for ALA/ALA
derivative detection according to Section 2. The percentage of ALA
derivatives recovered was calculated.
Table 3
ALA and ALA derivatives accumulation in cells
Total nanomoles ALA
or ALA derivativeaPercentage of ALAb
Control 2.24 ± 0.09 100c
ALA 18.06 ± 0.90 100
He-ALA 58.72 ± 8.58 20
Undecanoyl-ALA 4.36 ± 0.14 N.D.
THP-ALA 50.19 ± 6.49 40
a ALA or ALA derivatives were determined in cells according to the
procedure described in Section 2 after 3 h exposure to 0.6 mM ALA or
ALA derivatives. Controls correspond to basal levels of untreated
cells. N.D.: non-detectable by this method, without distinction be-
tween ALA and derivative.b ALA was distinguished from ALA derivatives employing the
sodium acetate eluates data. The percentage of free ALA was calcu-
lated from the total ALA+ALA derivatives data (see footnote a), after
subtracting PBG contribution.c Theoretical, N.D.
G. Di Venosa et al. / Journal of Photochemistry and Photobiology B: Biology 75 (2004) 157–163 161
Solutions of 2 and 3 M sodium acetate, 1 and 4 N HCl, 1
M BaCl2 and 5 M KOH only eluted 5–10% of the com-
pounds. When ammonium acetate was used, interfer-
ences in the colorimetric quantification of the eluates
were found.
Only after employing 10 M HCl, almost 100% of theHexyl, Undecanoyl and THP ALA esters was recovered
at all the concentrations tested (Table 2). TLC of the
dialyzed eluates confirmed that they contain mainly
ALA esters and discarded the hypothesis that the acidic
conditions could have provoked the hydrolysis and re-
lease of the derivatives bound to the resin. On the other
hand, only 70–80% of the Me-ALA ester was recovered
in the HCl fractions.At difference with ALA elution, the release of ALA es-
ters in the eluates was gradual and each 1 ml HCl of the
seven fractions collected, contained 10–20% of the total
amount of ALA ester applied into the column. The sen-
sitivity of the method was 5 times lower as compared to
ALA, because of the more even distribution of the esters
among the fractions eluted. In addition, taking into ac-
count the volume of NaOH required to raise the pH, thislimit was increased to 5 nmol/ml fraction. This leads to a
lower estimation of the recovery of 0.06 lmol of the
derivatives, and raises the limit of detection of the
ALA derivatives to a total of 60 nmol theoretically,
although taking into account the variation in the eluates,
we found that the limit was 150 nmol. When employing
60 nmol, we underestimate a 10% of the total.
We recovered only a 10% of the 6 lmol ALA in theHCl fraction, due to saturation of the resin under these
conditions.
3.3. Separation of ALA and ALA derivatives mixtures
Mixtures of equimolar concentrations of ALA and
ALA derivatives were passed through the Dowex 50
X8 resin (data not shown). A mixture of 6; 0.6 and0.06 lmol of ALA and an equal amount of the different
ALA derivatives were seeded on top of the column and
eluted with sodium acetate and HCl as described above.
The percentages of recovery of both ALA and ALA
derivatives were identical to the recoveries of the com-
pounds alone.
3.4. ALA, ALA derivatives and PBG determinations in
cells
Table 3 shows ALA and ALA derivatives accumula-
tion in cells treated with ALA or ALA derivatives, and
the percentage of ALA present in the free form.
We can clearly see that ALA and/or He-ALA accu-
mulated from He-ALA is 4 times higher than ALA
accumulated from ALA, but only 20% has been con-verted to the ALA free form. The amount of ALA
and/or THP-ALA accumulated from THP-ALA is 2.8
times higher than the amount accumulated from ALA
but again only 40% is converted into ALA.ALA and/or Undecanoyl-ALA accumulated from the
latter is very low and it could not be separated by ionic
chromatography.
Due to the lower sensitivity of the method for the
determination of ALA derivatives from the HCl eluates,
the percentage of free ALA was calculated considering
the sodium acetate eluates data and the total ALA after
subtracting the PBG contribution, rather than estimat-ing the ALA derivatives recovery in the HCl eluates.
The amount of PBG is very low for all the conditions
(data not shown); however, because PBG is not retained
in the Dowex 50 resin, there are not any possible inter-
ference in the ALA or ALA derivatives quantification of
the eluates
4. Discussion
We demonstrated that ALA derivatives can be quan-
tified by means of acetyl acetone condensation followed
by exposure to Ehrlich reagent, a method commonly
used for ALA determination in porphyric patients. The
absorbance spectra of the Ehrlich reaction products were
identical for both ALA and ALA derivatives, and fur-ther tests demonstrated that the ester was not hydrolized
during the condensation step or the Ehrlich reaction.
We have also described a simple and cheap method to
distinguish ALA from ALA derivatives based on ionic
exchange extraction.
ALA is retained in cationic columns through its
amine group and ALA derivatives should have been re-
tained by the same mechanism. However, we were sur-prised to find out that such a high ionic strength was
needed to release all the ALA derivatives from the resin.
We hypothesize that such an acidic condition is lead-
ing the resin to a transient structural relaxation which
162 G. Di Venosa et al. / Journal of Photochemistry and Photobiology B: Biology 75 (2004) 157–163
allows the release of the entrapped molecules. After
regeneration, the resin can be reused and conserves its
properties, showing that the changes driven by the ex-
treme acidic conditions were reversible.
Whereas ionic interactions are involved in the reten-
tion of all the ALA derivatives, other factors are likelyto be contributing in their release. The chain length is
apparently crucial in this process, since the shorter
methyl ALA derivative is partially retained (up to
70%) in the resin mesh. On the other hand, the cyclic
tetrahydropyranyl structure is also entrapped in the re-
sin to the same extent as the hexyl or undecanoyl chains.
When the same set of experiments was carried out in
batch instead of column, identical results were obtained.This discards the importance of the resin bead size or the
entrapment of the molecules among the beads. On the
contrary, we postulate that there occurs an interaction
with the divinyl benzene–polystyrene molecules. In this
regard, we have used the Dowex 50 X2, a resin with a
lower percentage of crosslinking and we have found that
Undecanoyl-ALA and He-ALA were identically re-
tained in the mesh. On the other hand, 100% of Me-ALA and 70% of THP-ALA were released in the sodium
acetate eluates. This increased release of the ALA esters
with cyclic or short chains in the sodium acetate frac-
tions reinforces the hypothesis that the ester chain
length is crucial in the resin interaction.
Employing cell lines we were able to distinguish be-
tween ALA and the derivatives He-ALA and THP-
ALA, and we found that only part of the esters areintracellularly being hydrolyzed to release ALA, show-
ing the limitations of the esterases in this process.
On the other hand, we were unable to use the proce-
dure to separate Me-ALA from ALA because its has a
shorter lipophilic chain and the elution profile overlaps
ALA release.
We were able to separate ALA from ALA derivatives
and quantify them in such amounts that this method canbe well applied to their determination in cell extracts. In
our particular cell system, where porphyrin synthesis
have plateaux values at 0.6 mM ALA or ALA deriva-
tives and 20–40% of the ALA esters are converted to
ALA, the amount of intracellular ALA esters is high en-
ough to be detected by this method. However, according
to current literature, different cell lines show different
ALA plateaux values. In addition, esterases activities de-pend on cells types and consequently, a higher amount
of cells may be eventually needed to adjust to the detec-
tion limit of our method.
5. Abbreviations
ALA 5-aminolevulinic acidDMAB dimethylaminobenzaldehyde
He-ALA ALA-hexyl ester
Me-ALA ALA-methyl ester
PBG porphobilinogen
PDT photodynamic therapy
THP-ALA R,S-ALA-2-(hydroximethyl)tetrahydro
pyranyl ester
PBS phosphate-buffered saline
Acknowledgements
This research was supported by grants from the Na-
tional Agency for Science and Technology (05-09042)
and the National Research Council (CONICET)(05508/02). A.M. del C.B., H.F. and A.C. hold the posts
of Superior, Associate and Assistant Researcher at the
CONICET respectively. G.D.V. is a CONICET fellow.
C.P. is a Wellcome Trust fellow.
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