Effect of pterin impurities on the fluorescence and photochemistry of commercial folic acid

Article abstract of DOI:10.1016/j.jphotobiol.2018.03.007

Folic acid, or pteroyl?L?glutamic acid (PteGlu) is a conjugated pterin derivative that is used in dietary supplementation as a source of folates, a group of compounds essential for a variety of physiological functions in humans. Photochemistry of PteGlu is important because folates are not synthesized by mammals, undergo photodegradation and their deficiency is related to many diseases. We have demonstrated that usual commercial PteGlu is unpurified with the unconjugated oxidized pterins 6?formylpterin (Fop) and 6?carboxypterin (Cap). These compounds are in such low amounts that a normal chromatographic control would not detect any pterinic contamination. However, the fluorescence of PteGlu solutions is due to the emission of Fop and Cap and the contribution of the PteGlu emission, much lower, is negligible. This is because the fluorescence quantum yield (Φ_F) of PteGlu is extremely weak compared to the Φ_F of Fop and Cap. Likewise, the PteGlu photodegradation upon UV-A radiation is an oxidation photosensitized by oxidized unconjugated pterins present in the solution, and not a process initiated by the direct absorption of photons by PteGlu. In brief, the fluorescence and photochemical properties of PteGlu solutions, prepared using commercially available solids, are due to their unconjugated pterins impurities and not to PteGlu itself. This fact calls into question many reported studies on fluorescence and photooxidation of this compound.

Full text of DOI:10.1016/j.jphotobiol.2018.03.007

JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Effect of pterin impurities on the fluorescence and photochemistry of
commercial folic acid
M. Laura Dántola, M. Noel Urrutia, Andrés H. Thomas⁎
Instituto de Investigaciones Fisicoquímicas Teóricas y Aplicadas (INIFTA), Departamento de Química, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, CCT
La Plata-CONICET, Casilla de Correo 16, Sucursal 4, 1900 La Plata, Argentina
A R T I C L E I N F O
A B S T R A C T
Keywords:
Folates
Photostability
UV-A radiation
Pterinic impurities
Photosensitization
Folic acid, or pteroyl‑L‑glutamic acid (PteGlu) is a conjugated pterin derivative that is used in dietary supple-
mentation as a source of folates, a group of compounds essential for a variety of physiological functions in
humans. Photochemistry of PteGlu is important because folates are not synthesized by mammals, undergo
photodegradation and their deficiency is related to many diseases. We have demonstrated that usual commercial
PteGlu is unpurified with the unconjugated oxidized pterins 6‑formylpterin (Fop) and 6‑carboxypterin (Cap).
These compounds are in such low amounts that a normal chromatographic control would not detect any pterinic
contamination. However, the fluorescence of PteGlu solutions is due to the emission of Fop and Cap and the
contribution of the PteGlu emission, much lower, is negligible. This is because the fluorescence quantum yield
(ΦF) of PteGlu is extremely weak compared to the ΦF of Fop and Cap. Likewise, the PteGlu photodegradation
upon UV-A radiation is an oxidation photosensitized by oxidized unconjugated pterins present in the solution,
and not a process initiated by the direct absorption of photons by PteGlu. In brief, the fluorescence and pho-
tochemical properties of PteGlu solutions, prepared using commercially available solids, are due to their un-
conjugated pterins impurities and not to PteGlu itself. This fact calls into question many reported studies on
fluorescence and photooxidation of this compound.
1. Introduction
problems [5–7]. Folate requirements increase in periods of rapid cell
division and growth, and, therefore, it is important that pregnant
Folic acid, or pteroyl‑L‑glutamic acid (PteGlu) is a conjugated pterin
derivative, with a chemical structure composed by three moieties: a
6‑methylpterin (Mep) residue, a p‑aminobenzoic acid (PABA) residue,
and a glutamic acid (Glu) residue (Scheme 1a). In living systems,
PteGlu is present in multiple forms including molecules attached to
several glutamate residues and dihydro and tetrahydro derivatives.
Folate is the generic term for this large family of chemically related
compounds.
Tetrahydrofolate and its derivatives act as coenzymes in one-carbon
transfer reactions required in the biosynthesis of nucleic acids and
proteins [1] and, in consequence, these compounds are essential for a
variety of physiological functions in humans. Moreover, a deficit of
folate leads to several diseases such as megaloblastic anemia [2], cor-
onary heart disease [3], neurological disorders [4], and fertility
women keep folate concentrations at an appropriate level [8]. Folate
deficiency in pregnancy is related to neural tube defects (NTD), such as
spina bifida and anencephaly [9,10].
Mammals do not synthesize folates and therefore they have to get
them from food. In some situations, like anemia or pregnancy, folate
supplementation is needed. PteGlu is inexpensive to produce, more
stable than most members of folate's family and efficiently metabolized
into biologically active derivatives, such as 5‑methyltetrahydrofolic
acid. Due to these properties, PteGlu is used in tablet form and in for-
tified foods for dietary supplementation [1,11].
The correlation between NTD and UV-A (315–400 nm) exposure has
been described in amphibian larvae [12] and in women who had used
artificial tanning sun beds during the first weeks of pregnancy [13]. In
addition, epidemiological data have shown that the prevalence of NTD
a,
Abbreviations: absorbance, A; absorbed photon flux density, qn,
V; acetonitrile, ACN; ammonium acetate, NH4Ac; 6‑carboxypterin, Cap; emission wavelength, λem; excitation
p
wavelength, λexc; folic acid, PteGlu; folic acid radical cation, PteGlu%+; formic acid, HCOOH; 6‑formylpterin, Fop; fluorescence, FL; fluorescence quantum yields, ΦF; glutamic acid, Glu;
high-performance liquid chromatography, HPLC; hydrogen peroxide, H2O2; incident photon flux density, qn, p0, V; incident photons per time interval, qn, p0; liquid chromatography/mass
spectrometry, LC/MS; 6‑methylpterin, Mep; neural tube defects, NTD; p‑aminobenzoic acid, PABA; p‑aminobenzoylglutamic acid, PABA-Glu; photosensitizers, Sens; photosensitizer
radical anion, Sens%−; reference fluorophore, R; retention times, tr; superoxide anion, O2%−; total fluorescence intensities, IF; triplet excited state of the sensitizer, 3Sens⁎; volume, V
⁎
Corresponding author at: C. C. 16, Sucursal 4, B1904DPI, La Plata, Argentina.
E-mail address: athomas@inifta.unlp.edu.ar (A.H. Thomas).
https://doi.org/10.1016/j.jphotobiol.2018.03.007
Received 27 December 2017; Received in revised form 19 February 2018; Accepted 5 March 2018
Availableonline09March2018
1011-1344/©2018PublishedbyElsevierB.V.

M.L. Dántola et al.
JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
Scheme 1. a) Photooxidation of PteGlu in air-equilibrated aqueous solutions under UV-A irradiation. b) Mechanism of the oxidation of PteGlu photosensitized by Fop. Similar processes
take place with other aromatic pterins acting as photosensitizers.
is higher in light-skinned people [14,15]. In 1978, Branda and Eaton
proposed that one of the main functions of skin pigmentation is to avoid
photolysis of folate [16]. This hypothesis was based on the following
facts: i) light-skinned patients undergoing photochemotherapy (i.e.,
psoralen plus UV-A) showed lower serum folate levels than healthy
controls, and ii) a 30–50% loss of folate in human plasma was observed
after in vitro exposure to simulated sunlight. Recent reports indicate
that both in vitro and in vivo exposure of human blood to UV-A radia-
tion, lead to photodegradation of folate [17,18].
PteGlu can be excited by solar radiation and artificial sources of
light since its absorption spectrum shows bands in the UV-B
(280–315 nm) and UV-A spectral regions (Fig. S1, Supplementary ma-
terial). The photodegradation of PteGlu was reported for the first time
in the late 1940s [19] and, since then, many studies on the photo-
chemistry of this compound, as a model of folates, have been conducted
in aqueous solutions and other media [20–28]. In the absence of
oxygen, PteGlu is photostable, but excitation in air-equilibrated solu-
tions leads to cleavage and oxidation of the molecule, yielding
6‑formylpterin (Fop) and p‑aminobenzoylglutamic acid (PABA-Glu) as
photoproducts (Scheme 1a). In turn, Fop is transformed into 6‑car-
boxypterin (Cap) upon further photooxidation.
rate of PteGlu consumption significantly increases. In 2010, we re-
ported that the photooxidation products of PteGlu are able to photo-
induce the degradation of this molecule (Scheme 1b) [29]. That means
that an “auto-photo-catalytic” effect is involved, where Fop photo-
sensitizes the oxidation of PteGlu, and that explains the acceleration in
the consumption of reactant when its products accumulate in the
medium. In general terms, the photosensitization consists in the che-
mical alteration occurring in one molecular entity as a result of the
initial absorption of radiation by another molecular entity called the
photosensitizer. This process, in which no excitation of PteGlu is
needed, also takes place with other pterins as photosensitizers (Sens),
thus revealing a general mechanism. After excitation of the Sens, the
first step of the process involves an electron transfer from the PABA unit
of PteGlu to the triplet excited state of the Sens (3Sens*) to form the
corresponding radical ions, the photosensitizer radical anion (Sens%−
)
and the PteGlu radical cation (PteGlu%+). The electron transfer from
Sens%− to O2 regenerates the Sens and forms superoxide anion (O2%−),
which disproportionates to form hydrogen peroxide (H2O2). Finally,
PteGlu degradation occurs as a result of the trapping of PteGlu%+ by
oxygen (Scheme 1b).
Although a large amount of reports about photodegradation of
PteGlu under UV-A radiation have been published, many questions
about the photochemistry of this compound remain without answer.
Upon UV-A irradiation the degradation of PteGlu in the presence of
oxygen is a sigmoidal function [22,24–26]: after a period of time the
158

M.L. Dántola et al.
JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
a,V
0,V
What is the mechanism of the initial step, that is, before the photo-
sensitized process dominates the degradation of PteGlu? Unconjugated
pterins are present as impurities in the commercial presentation of fo-
lates? If so, does the photosensitization by these compounds contribute
to the photodegradation of PteGlu? When the photophysical properties,
such as the fluorescence, of PteGlu are studied, do the unconjugated
pterins, present as impurities or generated photochemically, con-
tribute?
Motivated by the importance of the photochemistry of folates in
vivo, this work was aimed to answer the questions raised in the previous
paragraph. For this purpose, aqueous solutions of PteGlu were prepared
using different commercially available solids and were analyzed by
high-performance liquid chromatography (HPLC) and mass spectro-
metry to investigate pterinic impurities. PteGlu was purified by HPLC
and the photochemical and spectroscopic properties of solutions of
purified and unpurified PteGlu were compared.
q
= q (1 − 10−A
)
n,p
n,p
(1)
where A is the absorbance of the reactant at the excitation wavelength.
2.3. High-performance Liquid Chromatography (HPLC)
A Prominence equipment from Shimadzu (solvent delivery module
LC-20AT, on-line degasser DGU-20A5, communications bus module
CBM-20, auto sampler SIL-20A HT, column oven CTO-10AS VP, pho-
todiode array (PDA) detector SPD-M20A and fluorescence (FL) detector
RF-20A was employed for monitoring the photochemical processes and
to purified the PteGlu commercial sample. A Synergi Polar-RP column
(ether-linked phenyl phase with polar endcapping, 150 × 4.6 mm,
4 μm, Phenomenex) was used for isolation of PteGlu from HPLC runs
(preparative HPLC).·The column temperature was set at 25 °C and the
flow rate at 0.6 mL/min. Solution containing 100% NH4Ac (1 mM,
pH = 6.7
0.1) was used as mobile phase. The same column and runs
conditions were used to analyze the purity of the isolated PteGlu sample
and the progress of the photochemical processes.
2. Materials and Methods
2.1. General
2.4. Fluorescence Spectroscopy
2.1.1. Chemicals
Folic acid (PteGlu) was provided by Sigma Aldrich (solid I,
purity ≥ 97%) and Schircks Laboratories (solid II, purity > 98.5%).
Other pterins derivatives were purchased from Schircks Laboratories.
Ammonium acetate (NH4Ac) and formic acid (HCOOH) were purchased
from Sigma Chemical Co. Acetonitrile (ACN) was purchased from J. T.
Baker. Aqueous solutions were prepared using ultrapure water from
Milli-Q® purification system (Millipore Corporation, USA).
Steady-state and time-resolved fluorescence measurements were
performed on air-equilibrated aqueous solutions using a Single-Photon-
Counting equipment FL3 TCSPC-SP (Horiba Jobin Yvon). The equip-
ment has been previously described in detail [32].
In steady-state measurements the sample solution in a quartz cell
was irradiated with a 450 W Xenon source through an excitation
monochromator (FL-1004). The fluorescence, after passing through an
emission monochromator (iHR320), was registered at 90° with respect
to the incident beam using a room-temperature R928P detector. The
emission measurements were performed at 25 °C. Corrected fluores-
cence spectra obtained by excitation at 340 nm were recorded in the
range 375–580 nm. The total fluorescence intensities (IF) were calcu-
lated by integration of the fluorescence band centered at ca. 440 nm.
The fluorescence quantum yields (ΦF) were determined from the
corrected fluorescence spectra using the following equation:
2.1.2. Measurements of pH
A pH-meter sensION+pH31 GLP combined with a pH electrode
5010T (Hach) or microelectrode XC161 (Radiometer Analytical) were
used. The pH of the aqueous solutions was adjusted by adding drops of
0.1–0.2 M aqueous NaOH or HCl solutions with a micropipette. The
concentration of the acid and the base used for this purpose ranged
from 0.1 M to 2 M.
R IAR
ΦF = ΦF
IRA
(2)
2.1.3. UV/vis Spectrophotometric Analysis
Electronic absorption spectra were recorded at room temperature
on a Shimadzu UV-1800. Measurements were made using quartz cells of
0.4 and 1.0 cm optical path length.
where I is the integrated intensity, A is the absorbance at the excitation
wavelength and the superscript R refers to the reference fluorophore. In
our experiments, pterin in acid medium (pH = 6.0) was used as a re-
ference (ΦF = 0.32) [33]. The sample and reference were excited at the
same wavelength. To avoid inner filter effects, the absorbance of the
solutions at the excitation wavelength was kept below 0.10.
In time-resolved experiments a NanoLED source (maximum at
341 nm) was used for excitation and the emitted photons, after passing
through a monochromator, were detected by a TBX-04 detector and
counted by a FluoroHub-B module.
2.2. Steady-state Irradiation
2.2.1. Irradiation Setup
Air equilibrated aqueous solutions containing PteGlu were irra-
diated in quartz cells (1.0 cm optical path length) at room temperature
using a Rayonet RPR lamp (Southern N. E. Ultraviolet Co.) with emis-
sion centered at 350 nm (bandwidth ~ 20 nm).
2.5. Mass Spectrometry Analysis
2.2.2. Actinometry
Aberchrome 540 (Aberchromics Ltd.) was used as an actinometer
The liquid chromatography mass spectrometry system was equipped
with an UPLC chromatograph (ACQUITY UPLC from Waters) coupled to
a quadrupole time-of-flight mass spectrometer (Xevo G2-QTof-MS from
Waters) (UPLC-QTof-MS). UPLC analyses were performed using the
Acquity UPLC BEH Phenyl (1.7 μm, 2.1 × 50 mm) column (Waters). An
isocratic elution with 90% of aqueous HCOOH (0.1% v/v) and 10% of
ACN was used as mobile phase with a flow rate of 0.2 mL/min. Mass
chromatograms, i.e. representations of mass spectrometry data as
chromatograms (the x-axis representing time and the y-axis signal in-
tensity), were registered using different scan ranges. The mass spec-
trometer was operated in both positive (ESI+) and negative (ESI−) ion
modes.
0,V
for the measurement of the incident photon flux density (q ) at the
n,p
excitation wavelength, which is the amount of incident photons per
time interval (qn0,p) and divided by the volume (V) of the sample [30].
Aberchrome 540 is the anhydride form of the (E)‑α‑(2,5‑di-
methyl‑3‑furylethylidene)(isopropylidene)succinic acid which, under
irradiation in the spectral range 316–366 nm leads to a cyclized form.
0,V
The method for the determination of q
has been described in detail
n,p
[31].
The value of q
was 3.7 ( 0.4) × 10−6 Einstein L−1 s−1. The
0,V
n,p
a,V
value of the absorbed photon flux density (
q
) was calculated from
n,p
0,V
q
according to the Lambert-Beer law:
n,p
159

M.L. Dántola et al.
JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
Table 1
Values of m/z recorded for the molecular ions in MS spectra of the compounds present in a solution of PteGlu (100 μM) prepared using solid I.
Compound
Elemental composition [M]
ESI+ [M+H]+
ESI− [M−H]−
Observed m/z
Calculated m/z
Error (ppm)
Observed m/z
Calculated m/z
Error (ppm)
PteGlu
PABA-Glu
Fop
C19H19N7O6
442.1472
267.0981
192.0529
208.0478
442.1470
267.0975
192.0516
208.0465
0.45
2.25
6.77
6.25
440.1324
265.0823
190.0367
206.0323
440.1324
265.0830
190.0370
206.0320
0.00
C
12H14N2O5
−2.64
−1.58
1.46
C7H5N5O2
C7H5N5O3
Cap
3. Results and Discussion
3.1. Identification of the Impurities Present in Commercial PteGlu
In addition, the percentage of each impurity was determined in both
commercial solids. For this purpose, equal masses of solid I and II, were
dissolved in the same volume of H2O (pH = 6.0
0.1) in dark con-
ditions. After that, the same volume of each solution was analyzed by
HPLC. The amount of each impurity was determined by integration of
the peaks of the chromatograms recorded with the FL-detector, using
the calibration curves of each compound. The values obtained are
presented in Table 2.
Results presented in this section showed that usual commercial
PteGlu is unpurified with unconjugated oxidized pterins. These com-
pounds are in such low amounts that they are not detected by HPLC
using spectrophotometric detection, which is the most usual in chro-
matography. In consequence a normal chromatographic control of
commercial PteGlu would not detect any pterinic contamination.
Most of the PteGlu commercial solids used for photochemical stu-
dies are of suitable purity and are used without further purification.
Nevertheless little is known about the chemical nature of the im-
purities. To the best of our knowledge, only one work has investigated
this issue and Cap and PABA-Glu have been reported as impurities of
commercial PteGlu [34].
In order to further investigate the impurities, aqueous solutions of
PteGlu were prepared (pH = 6.0
0.1) from commercial solids of
different manufacturers (Materials and Methods section) in the absence
of light and the samples were analyzed by liquid chromatography/mass
spectrometry (LC/MS), using a UPLC equipment coupled to a mass
spectrometer (UPLC-QTof-MS, Materials and Methods section). The
chromatograms of solutions recorded using the UV/vis detector,
showed only one peak at retention times (tr) of 2.10 min (Fig. 2S,
Supplementary material). Analyzes were performed in both positive
and negative ion modes (ESI+ and ESI−, respectively). For the peak at tr
2.10 min, the signals corresponding to the intact molecular ion of
PteGlu as [M+H]+ and [M−H]−species were observed (M = PteGlu)
(Table 1). The mass spectra recorded in both ESI+ and ESI− modes for
different tr values revealed the presence of several compounds (Figs. 3S
and 4S, Supplementary material). As shown in Table 1, three of the ions
detected in the mass spectra clearly correspond to Fop, Cap and PABA-
Glu.
The samples were then analyzed by HPLC coupled to a PDA-detector
(Materials and Methods section). 75 μL of a solution of PteGlu (20 μM)
prepared from solid I were injected and chromatograms at different
analysis wavelengths were recorded, all of them showing only one peak
at tr 4.9 min (Fig. 1a). The spectrum registered for this peak corre-
sponded to the spectrum of PteGlu solutions recorded in a spectro-
photometer. This first analysis suggested a suitable purity of the sample.
However, when more concentrated solutions were injected new peaks
with low intensity appeared (inset Fig. 1a), revealing the presence of
several impurities at low concentrations. Absorption spectra were re-
corded for these impurities and two of them at tr values of 1.9 and
12.5 min were compatible with spectra of PABA-Glu and Fop, respec-
tively (inset Fig. 1a).
3.2. Purification of PteGlu
To assess the effect of the oxidized pterins, present as impurities, on
the spectroscopic and photochemical properties of the PteGlu aqueous
25
20
15
10
5
tr= 1.9 min
(PABA-Glu)
200
150
100
50
PteGlu
a)
20
10
0
2
tr= 12.5 min
(Fop)
1
0
200 250 300 350 400 450
(nm)
0
2
4
6
8
10
12
14
Retention time (min)
0
14
12
10
8
b)
Cap
20
15
10
5
PABA-Glu
0
0
1
2
3
4
5
6
7
Retention time (min)
6
The solutions were also analyzed by HPLC using the FL-detector
(Materials and Methods section). The conditions for the analysis were
chosen taking into account the fluorescence properties of unconjugated
pterins [33]: excitation wavelength (λexc) = 350 nm, emission wave-
length (λem) = 450 nm. The chromatograms registered under these
conditions showed 4 and 2 peaks for the solutions prepared with solid I
and II, respectively (Fig. 1). The comparison of the tr values observed in
these chromatograms with those obtained with a series of solutions of
standards of unconjugated pterins revealed that two of the impurities
are Cap and Fop. In another set of analyses, taking into account the
fluorescent properties of PABA-Glu [35], the optimal conditions for the
detection of this compound were set: λexc = 270 nm, λem = 350 nm.
The chromatograms registered under these conditions (inset Fig. 1b)
showed a peak with the same tr value as that observed for a standard
solution of PABA-Glu.
4
Fop
2
0
0
5
10
15
20
25
Retention time (min)
Fig. 1. HPLC analysis of aqueous solution of PteGlu. a) Chromatograms of solutions
prepared from solid I using the PDA detector at 270 nm. Main graph: chromatogram
recorded after injecting 75 μL of a solution 20 μM; inset: chromatogram recorded after
injecting 75 μL of a solution 240 μM and absorption spectra of the peaks with tr 1.9 and
12.5 min. b) Chromatograms obtained using the FL detector of solutions prepared from
solid
exc = 350 nm, λem = 450 nm; inset: λexc = 270 nm, λem = 350 nm. [PteGlu] = 20 μM,
pH = 6.0.
I (solid black line), solid II (dashed line) and PteGlu purified (gray line);
λ
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M.L. Dántola et al.
JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
Table 2
fluorescence of the solutions of PteGlu, the peaks of the chromatograms
recorded using the FL-detector (Fig. 1) were integrated. The percentage
of the area of each peak corresponds to the percentage of the fluores-
cence of the solution emitted by each compound under the conditions
chosen for the detection (λexc = 350 nm, λem = 450 nm). As shown in
Table 3, even in the solution of purified PteGlu, the compounds re-
sponsible for the fluorescence of the solution upon excitation at 350 nm
are Fop and Cap. It is worth mentioning that in the case of the solutions
prepared from solid I, the HPLC analysis showed the presence of other
fluorescent compounds different from Fop and Cap that also contribute
to the emission of the solution upon excitation with UV-A radiation.
In time-resolved experiments, fluorescence decays were analyzed
for the different solutions studied. The solutions prepared from solid II
and the purified PteGlu were fitted with three exponential functions
and the obtained lifetimes values (τF) with their corresponding relative
intensities are shown in Table 3. For both samples the two most intense
components presented τF values (τF2 and τF3) very close to those pre-
viously reported for Cap and Fop, respectively (τF(Fop) = 7.9 ( 0.4) ns,
Amount of each impurity (expressed in mol) per 100 mol of PteGlu in solutions prepared
from different commercial solids (Materials and Methods section) and with purified
PteGlu.
PteGlu solution
PABA-Glu
Cap
Fop
Solid I
Solid II
Purified PteGlu
1.0
0.7
0.3
0.2
0.2
0.1
0.17
0.16
0.014
0.01
0.02
0.006
1.3
0.7
0.2
0.1
0.2
0.1
solutions prepared from commercial solids, PteGlu was isolated from
HPLC runs (Materials and Methods section). We decided to purify the
PteGlu present in solutions prepared from solid II since it is the purest
one. PteGlu was collected using NH4Ac aqueous solution (1 mM,
pH = 6.7
adjusted at 6.0
0.1) as an eluent, the pH of the resulting solution was
0.1 and the absorption spectrum was controlled.
The HPLC analysis of the collected fraction suggested that the iso-
lation of PteGlu reduced significantly the amount of each impurity
present in the sample (Fig. 1, Table 2), but they could not be totally
eliminated. It is noteworthy that while the relative concentration of Cap
decreased to less than 10% of the initial one, the elimination of Fop
upon purification was much less efficient (Table 2).
τF(Cap) = 5.8 ( 0.4) ns) [33]. In addition, the relative fluorescence
intensity of the two components is quite similar to the relative emission
of Fop and Cap calculated in the corresponding solutions from chro-
matograms recorded using the FL-detector (Table 3). Finally, it is worth
mentioning that kinetic analysis of the decays recorded for solutions
prepared from solid I could not be carried out fitting with up to four
exponential functions, which is not surprising due to the considerable
presence of other fluorescent impurities in addition to Fop and Cap
(Table 3).
Therefore, the time-resolved study performed with solutions pre-
pared from solid II and the purified PteGlu, in agreement with fluor-
escence steady-state experiments and HPLC-FL analysis, indicate that
fluorescence of Fop dominates the emission of the analyzed samples
and Cap also contributes. The third fluorescent detected component
(τF1) might correspond to PteGlu, but more studies should be done to
determine correctly its τF value.
3.3. Fluorescence Properties
The fluorescence properties of the solutions of PteGlu purified and
unpurified were studied and compared. Corrected fluorescence spectra
of the three solutions obtained by excitation at 340 nm were recorded in
the range of 375–580 nm, under the same experimental conditions
(absorbance at the excitation wavelength (λexc), pH 6.0, slits of the
excitation and emission monochromators, etc.). As shown in Fig. 2, the
more unpurified is the sample, the more intense is the emission spectra.
Moreover, the apparent fluorescence quantum yields (ΦF) were calcu-
lated using Ptr in aqueous solution (pH 5.5) as
a reference
(ΦF = 0.32 0.01) [33], and the value of this parameter decreased
The results presented in this section showed that the fluorescence of
a solution of PteGlu prepared using commercially available solids
cannot be attributed to PteGlu and the fluorescence of Fop and Cap
present as impurities is what really is measured. This fact occurs be-
cause the real fluorescence of PteGlu is extremely weak. The ΦF value of
PteGlu is negligible compared to those of Fop and Cap, therefore al-
though these compounds are in very small concentrations in the solu-
tion, their fluorescence dominates the emission of the PteGlu solution.
These findings suggest that many works reporting data about the
fluorescence of PteGlu should be revised.
with the concentration of Fop and Cap in the sample (Table 3). These
results indicate that the fluorescent emission of a solution of PteGlu is
governed by the unconjugated pterins present as impurities and not by
the PteGlu itself.
To quantify the contribution of Fop and Cap to the total
3.5
3.0
2.5
2.0
1.5
1.0
0.5
3.4. Stability of PteGlu Under UV-A Radiation
Many reports have studied the photodegradation of PteGlu in aqu-
eous solutions under UV-A radiation (see Introduction). Nevertheless,
no one has investigated the effect of the pterinic impurities on the
photostability of PteGlu. Therefore, air equilibrated aqueous solutions
prepared from solids I and II and purified PteGlu, were exposed to UV-A
irradiation for different periods of time. The irradiated samples were
analyzed by HPLC and the concentration profiles of PteGlu, Fop and
Cap were obtained.
The initial rate of PteGlu consumption (−(d[PteGlu]/dt)0) was
calculated from the corresponding plots of PteGlu concentration as a
function of irradiation time (Fig. 3a) and values of 1.1 ( 0.1)
μM min−1, 0.41 ( 0.04) μM min−1 and 0.046 ( 0.008) μM min−1
were obtained for solutions prepared from solids I and II and purified
PteGlu, respectively. It is clear that (−d[PteGlu]/dt)0 increased with
the concentration of pterinic impurities, thus indicating that initial
consumption of PteGlu depends on the presence of Fop and Cap in the
medium. A similar behavior was observed for the formation of Fop
(Fig. 3b), that is, the higher the purity of the solution, the lower the rate
of Fop formation. These results suggest that PteGlu is not degraded by
0.0
375 400 425 450 475 500 525 550 575
(nm)
Fig. 2. Corrected fluorescence spectra (λexc = 340 nm) of an aqueous solution of purified
(gray solid line) and unpurified folic acid obtained from solid I (black solid line) and solid
II (black dashed line); the absorbance at λexc was 0.1 for all the samples, pH = 6.0
0.1.
161

M.L. Dántola et al.
JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
Table 3
Apparent fluorescence quantum yields (ΦF) and fluorescence lifetime (τF) calculated for solutions prepared from solid I, solid II and purified PteGlu (λexc = 350 nm, pH = 6.0); Relative
Emission calculated from chromatograms recorded using the FL-detector (Fig. 1, λexc = 350 nm, λem = 450 nm).
PteGlu solution
ΦF (10−4
)
τF1 (ns)
τF2 (ns)
τF3 (ns)
Relative emission (%)
PteGlu
Cap
Fop
Others
20
Solid I
Solid II
Purified PteGlu
51
21
7
5
2
–
–
–
2.3
4
12.1
0.6
11
22
16
2
5
3
67
67
73
8
7
7
6
0.5 (5%)
0.5 (10%)
4.3 (20%)
3.9 (20%)
8.8 (75%)
8.6 (70%)
1
8
2
1
0.7
–
35
34
33
32
60
50
40
30
20
10
0
a)
b)
a)
b)
12
9
1.5
1.0
0.5
0.0
6
3
0
c)
20
15
10
5
0.0
0.4
0.8
1.2
1.6
2.0
2.4
irradiation time (min)
Fig. 3. Time-evolution of a) PteGlu and b) Fop concentrations during irradiation of air-
equilibrated aqueous solution of purified PteGlu (●), unpurified PteGlu obtained from
commercial solid I (■) and II (▼). pH = 6.0
0.1.
0
0
2
4
6
8
10
12
14
direct excitation of its pterinic moiety, but by photosensitization with
unconjugated pterins present in the solutions as impurities. A similar
behavior was also observed in other pterin derivatives, such as dihydro
and tetrahydropterins. In this case, the reduced pterins suffer auto-
oxidation yielding aromatic pterins as products, which are able to
sensitize the photodegradation of reduced pterin [36].
irradiation time (min)
Fig. 4. Time-evolution of PteGlu, Fop and Cap concentrations during irradiation of an air
equilibrated aqueous solution of purified PteGlu (●), unpurified PteGlu obtained from
commercial solid I (■) and II (▼). pH = 6.0
0.1.
The behavior observed for longer irradiation times (Fig. 4) was in
agreement with previous studies, that is, after a period of time the rate
of PteGlu degradation increased with time [22,24] due to the accu-
mulation of products (oxidized unconjugated pterins) that are able to
photosensitize PteGlu. Although this behavior was observed for the
three samples analyzed, the acceleration in the consumption of PteGlu
occurred at different irradiation times: the more unpurified the sample,
the shorter the time for the acceleration.
Therefore the rate of the overall photodegradation of PteGlu upon
UV-A radiation depends on the initial concentration of oxidized pterins
present in the solution. The results shown in this section suggested that
when the photolysis of PteGlu is studied using aqueous solutions pre-
pared from commercially available solids, as a matter of fact, the
photodegradation of this compound initiated by the direct absorption of
photons is not investigated. Instead of that, what is really analyzed is
the degradation of PteGlu photosensitized by oxidized unconjugated
pterins.
4. Conclusions
Usual commercial PteGlu is unpurified with unconjugated oxidized
pterins, in particular, 6‑formylpterin (Fop) and 6‑carboxypterin (Cap).
These compounds are in such low amounts that they are not detected by
HPLC using spectrophotometric detection, which is the most usual in
chromatography to control purity. PteGlu was isolated from HPLC runs
and the concentration of the impurities was reduced significantly. The
fluorescence and photochemical properties of the PteGlu aqueous so-
lutions prepared from commercial solids and the purified solutions
were compared to assess the effect of the oxidized pterins, present as
impurities.
The fluorescence of PteGlu solutions is due to the emission of Fop
and Cap and the contribution of the PteGlu emission is negligible. This
is because the fluorescence quantum yield (ΦF) of PteGlu is extremely
weak compared to the ΦF of Fop and Cap. Likewise, the PteGlu
162

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JournalofPhotochemistry&Photobiology,B:Biology181(2018)157–163
photodegradation upon UV-A radiation is an oxidation reaction pho-
tosensitized by oxidized unconjugated pterins present in the solution,
and not a process initiated by the direct absorption of photons by
PteGlu.
The results presented have important implications because the
fluorescence and photochemical properties of PteGlu solutions, pre-
pared using commercially available solids, are dominated by the un-
conjugated pterins present as impurities and not by PteGlu itself. This
fact calls into question many reported studies on fluorescence and
photooxidation of this compound. Moreover, the photodegradation of
PteGlu in biological systems might be consequence of photosensitized
processes and the direct excitation of PteGlu might play a minor role.
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The present work was partially supported by Consejo Nacional de
Investigaciones Científicas y Técnicas (CONICET; Grant PIP 0304),
Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT;
Grants PICT 2012-0508 and PICT 2015-1988), and Universidad
Nacional de La Plata (UNLP; Grant X586 and X712). M.N.U. thanks
CONICET for postdoctoral research fellowship. A.H.T. and M.L.D. are
research members of CONICET. The authors thank Dr. Maira Gaspar
Tosato (INIFTA, CONICET) and Nathalie Martins-Froment of the
Service Commun de Spectrométrie de Masse (FR2599), Université de
Toulouse III (Paul Sabatier) for their crucial contributions in mass
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