Formation of 7-carboxyheptyl radical induced by singlet oxygen in the reaction mixture of oleic acid, riboflavin and ferrous ion under the UVA irradiation.

Identification of the radicals was performed for the standard reaction mixtures, which contained 4.3 mM oleic acid, 25 µM riboflavin, 1 mM FeSO(4)(NH(4))(2)SO(4), 10 mM cholic acid, 40 mM phosphate buffer (pH 7.4) and 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone under the UVA irradiation (365 nm), using an electron spin resonance, an high performance liquid chromatography-electron spin resonance and an high performance liquid chromatography-electron spin resonance-mass spectrometry. The electron spin resonance and high performance liquid chromatography-electron spin resonance measurements of the standard reaction mixtures showed prominent signals (α(N) = 1.58 mT and α(H)β = 0.26 mT) and peaks 1 and 3 (retention times, 37.0 min and 49.0 min). Since the peak 3 was not observed for the standard reaction mixture without oleic acid, the radical of the peak 3 seems to be derived from oleic acid. Singlet oxygens seem to participate in the formation of the oleic acid-derived radicals because the peak height of the peak 3 observed in the standard reaction mixture of D(2)O increased to 308 ± 72% of the control. The high performance liquid chromatography-electron spin resonance-mass spectrometry analysis showed that 7-carboxyheptyl radical forms in the standard reaction mixture.

Introduction

Of the various oxidative stresses, UV irradiation is one of the primary factors. Chronic exposure of solar UV irradiations to mammalian skins induces a number of biological responses, including erythema, edema, sunburn cell formation, hyperplastic responses, photoaging and skin cancer development.( Increasing evidences showed that free radicals may be involved in the acute sunburn reactions.( Indeed, reactive oxygen speceis (ROS) such as hydrogen peroxide, 1O2, O2−• and nitric oxide were detected under the UV irradiation.( Following UV-exposure, the ROS play a major role, in addition to DNA damage,( in producing lipid-derived radicals and the peroxidation of membrane lipids that seem to be responsible for the destruction of the cell membrane and ultimately the cell.( The UV-induced lipid peroxidation occurs either through a free radical chain reaction( or alternatively through a non-radical pathway by a direct reaction with singlet oxygen (1O2).(

Since UVB (315–280 nm) radiation constitutes only 5% of the solar UV radiation that reaches the surface of the earth, skin damages are not caused entirely by the UVB. Sufficient evidences indicated that UVA (400–315 nm) radiation, which accounts for the major portion of the solar UV radiation leads to skin damages.( The UVA-induced damages would be mediated by endogenous and/or exogenous photosensitizers. A particularly interesting photosensitizer is riboflavin (Rf), a member of the vitamin B2 complex, which is a natural compound present in most living organisms.( The Rf can potentially act as a natural endogenous photosensitizer.( The usual mechanism of action of this sensitizer is rather complex. The Rf photodecomposes under aerobic UV or visible irradiation, in most of the cases with the concurrent involvement of ROS such as singlet oxygen (1O2) and superoxide anions (O2−•).(

In spite of the intensive ESR studies on the reaction of Rf with natural or external compounds under the UVA irradiation, the radical species formed in the reaction mixtures have not been identified. In order to clarify the mechanism of UVA-induced skin damages mediated by endogenous and/or exogenous photosensitizers, in this study, identification of the lipid-derived radicals was performed for the standard reaction mixture which contained 4.3 mM oleic acid, 25 µM riboflavin, 1 mM FeSO4(NH4)2SO4, 10 mM cholic acid, 40 mM phosphate buffer (pH 7.4), and 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone under the UVA irradiation (365 nm) using the ESR, the HPLC-ESR and the HPLC-ESR-MS.( A spin trap reagent, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was used to trap the lipid-derived radicals.

Materials and Methods

Chemicals

α-(4-Pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was purchased from Tokyo Kasei Kogyo, Ltd. (Tokyo, Japan). Sodium azide and Rf were purchased from Wako Pure Chemical Industries (Osaka, Japan). Superoxide dismutase (SOD) from bovine erythrocytes and catalase from bovine liver were from Sigma-Aldrich Co. (St. Louise, MO). All other chemicals used were of analytical grade.

ESR studies

The ESR experiments were carried out on a JES-FR 30 Free Radical Monitor (JEOL Ltd., Tokyo, Japan). Operating conditions of the ESR spectrometer were: power, 4 mW; modulation width, 0.1 mT; time constant, 0.3 sec. Magnetic fields were calculated by the splitting of MnO (ΔH3–4 = 8.69 mT).

HPLC-ESR chromatography

An HPLC used in the HPLC-ESR consisted of a model 7125 injector (Reodyne, Cotati, CA), a model L-7100 pump (Hitachi Ltd., Ibaragi, Japan). A semi-preparative column (300 mm long × 10 mm i.d.) packed with TSKgel ODS-120T (TOSOH Co., Tokyo, Japan) was used. Flow rate was 2.0 ml/min throughout the HPLC-ESR experiments. For the HPLC-ESR, two solvents were used: solvent A, 50 mM acetic acid; solvent B, 50 mM acetic acid/acetonitrile (20:80, v/v). A following combination of isocratic and linear gradient was used: 0–40 min, 100% A to 20% A (linear gradient); 40–75 min, 80% B (isocratic). The eluent was introduced into a model JES-FR30 Free Radical Monitor (JEOL Ltd., Tokyo, Japan). The ESR spectrometer was connected to the HPLC with a Teflon tube, which passed through the center of the ESR cavity. The operating conditions of the ESR spectrometer were: power, 4 mW; modulation width, 0.2 mT; time constant, 1 sec. The magnetic field was fixed at the third peak in the double-triplet ESR spectrum (αN = 1.58 mT and αHβ = 0.26 mT) of the 4-POBN radical adducts (Fig. 1).

Fig. 1

ESR spectra of the standard reaction mixtures of oleic acid, Rf and ferrous ions under the UVA irradiation. The reaction and ESR conditions were as described in Materials and Methods. (A) An ESR spectrum of the standard reaction mixture. (B) An ESR spectrum of the standard reaction mixture without light. (C) An ESR spectrum of the standard reaction mixture without Fe2+. (D) An ESR spectrum of the standard reaction mixture without Rf. (E) An ESR spectrum of the standard reaction mixture without oleic acid.

HPLC-ESR-MS chromatography

The HPLC and ESR conditions were as described in the HPLC-ESR. The mass spectrometer (MS) used in the HPLC-ESR-MS was a model M-1200 HS electrospray ionization (ESI)-MS (Hitachi Ltd., Ibaragi, Japan). The operating conditions of the ESI-MS were: nebulizer, 180°C; aperture1, 120°C; N2 controller pressure, 19.6 N/cm2; drift voltage, 70 V; multiplier, 2000 V; needle voltage, 4000 V; polarity, positive; resolution, 48. The mass spectra were obtained by introducing the eluent from the ESR detector into the ESI-MS system just before the peak was eluted. The flow kept at 50 µl/min while the eluent was introducing into the ESI-MS.

Standard reaction conditions

The standard reaction mixture contained 4.3 mM oleic acid, 25 µM Rf, 40 mM phosphate buffer (pH 7.4), 10 mM cholic acid, 0.1 M 4-POBN and 1 mM FeSO4(NH4)2SO4 in a quartz test tube (100 mm long × 8 mm i.d.). The standard reaction mixtures without 1 mM FeSO4(NH4)2SO4 and 0.1 M 4-POBN were exposed to 7.8 J/cm2 UVA light under air using a 400 W UV lamp and a LXO365 bandpass filter (365 nm) (ASAHI SPECTRA Co., Tokyo, Japan). After the UVA irradiation, 0.1 M 4-POBN was added. The reactions were started by adding 1 mM FeSO4(NH4)2SO4 and performed at 25°C for 1 min. The reaction mixtures were aspirated into a Teflon tube centered in an ESR microwave cavity. And then, ESR spectra were measured.

Reaction in D2O

After water was removed from the standard reaction mixture without 0.1 M 4-POBN and 1 mM FeSO4(NH4)2SO4 using centrifugal concentrator cc-105 (Tomy Seiko Co., Ltd., Tokyo, Japan), D2O was added to the reaction mixture. The other reaction conditions were as described for the standard reaction conditions. The control reaction was performed by addition of H2O instead of D2O.

Results

ESR measurements of the standard reaction mixtures under the UVA irradiation

ESR spectra were measured for the standard reaction mixtures under the UVA irradiation (Fig. 1). A prominent ESR spectrum (αN = 1.58 mT and αHβ = 0.26 mT) was observed in the standard reaction mixture (Fig. 1A), suggesting that free radicals form in the standard reaction mixture. The ESR spectrum was hardly observed for the standard reaction mixture without the UVA irradiation (or Fe2+, or Rf, or oleic acid) (Figs. 1 B–E).

UVA irradiation energy dependence of the ESR peak height

UVA irradiation energy dependence of the ESR peak height was observed (Fig. 2). The ESR peak height increased with increase of the irradiation energy, and reached a plateau at 2 J/cm2.

Fig. 2

Irradiation energy dependence of the ESR peak heights. The reaction and ESR conditions were as described in Materials and Methods except for the irradiation energies.

HPLC-ESR analyses of the standard reaction mixtures under the UVA irradiation

To know which radicals formed in the standard reaction mixtures of oleic acid, Rf and iron ions under the UVA irradiation, the HPLC-ESR analyses were performed for the standard reaction mixtures at a modulation of 0.2 mT. Prominent peaks (peaks 1 and 3) were observed on the HPLC-ESR elution profile of the standard reaction mixture under the UVA irradiation (Fig. 3A). The retention times of the peaks 1 and 3 are 37.0 and 49.0 min, respectively. The peak 3 was hardly observed for the standard reaction mixture without the UVA irradiation (or Fe2+, or Rf, or oleic acid) (Figs. 3 B–E). The peak 1 was not observed for the standard reaction mixture without Rf (Fig. 3D).

Fig. 3

HPLC-ESR analyses of the standard reaction mixtures. The reaction and HPLC-ESR conditions were as described in Materials and Methods. (A) The standard reaction mixture. (B) The standard reaction mixture without light. (C) The standard reaction mixture without Fe2+. (D) The standard reaction mixture without Rf. (E) The standard reaction mixture without oleic acid.

HPLC-ESR-MS analyses of the standard reaction mixtures under the UVA irradiation

To determine the structures of the peak 1 and peak 3 compounds observed on the HPLC-ESR elution profile of the standard reaction mixture under the UVA irradiation, the HPLC-ESR-MS analyses were performed at a modulation of 0.2 mT. The HPLC-ESR-MS analysis of the peak 1 compound gave ions at m/z 377 (Fig. 4A). The ions at m/z 377 correspond to the protonated molecules of Rf, [M + H]+. ESR spectra of the peak 1 were measured (Fig. 5). Although no ESR signal was observed at a modulation of 0.1 mT (Fig. 5A), a broad ESR signal was observed at a modulation of 0.2 mT (Fig. 5B), suggesting that a relatively stable Rf radical with broad ESR signals forms in the reaction mixture.

Fig. 4

HPLC-ESR-MS analyses of the peaks 1 and 3. The reaction and HPLC-ESR-MS conditions were as described in Materials and Methods. (A) Peak 1. (B) Peak 3.

Fig. 5

ESR spectra of the peak 1. The reaction, ESR and HPLC-ESR conditions were as described in Materials and Methods except for the modulation values. (A) 0.1 mT. (B) 0.2 mT.

The HPLC-ESR-MS analysis of the peak 3 compound gave ions at m/z 251 and m/z 338 (Fig. 4B). The ions at m/z 338 correspond to the protonated molecules of the 4-POBN/7-carboxyheptyl radical adducts, [M + H]+. The fragment ions at m/z 251 correspond to the loss of (CH3)3C(O)N from the protonated molecules.

Effects of some compounds on the ESR and HPLC-ESR peak heights

To know whether singlet oxygens are involved in the radical formation, the reactions were performed in D2O. The ESR peak heights which were observed for the reaction in D2O increased to 248 ± 9% of the control (Table 1). The HPLC-ESR peak height of the peak 3 which were observed for the reaction in D2O also increased to 308 ± 72% of the control.

Table 1

Effects of some compounds on the ESR peak heights (ESR) and peak 3 peak heights (HPLC-ESR)

compounds added and irradiation energies	% control
D2O (ESR), 7.8 J/cm2	248 ± 9
D2O (HPLC-ESR), 7.8 J/cm2	308 ± 72
Azide (20 mM)(ESR), 7.8 J/cm2	198 ± 14
Azide (20 mM)(ESR), 1.95 J/cm2	156 ± 26
Azide (20 mM)(ESR), 1.95 J/cm2	149 ± 3*
Azide (0.2 mM)(ESR), 1.95 J/cm2	157 ± 27
SOD (300 units/ml)(ESR), 1.95 J/cm2	95 ± 5.5
Catalase (1260 units/ml)(ESR), 1.95 J/cm2	115 ± 23

Results are means ± SD of three experiments. The reactions were performed as described in Materials and Methods excect for the data indicated by *. The data indicated by * were obtained under the same conditons as Materials and Methods except for the concentration of phosphate buffer. The concentration of phosphate buffer was 140 mM.

When a singlet oxygen scavenger, azide was added to the standard reaction mixtures of oleic acid, the ESR peak height interestingly increased to 198 ± 14% (156 ± 26%) of the control under the 7.8 J/cm2 (1.95 J/cm2) UVA irradiation (Table 1). To check the pH change due to the addition of 20 mM sodium azide, the reactions were performed in the 140 mM phosphate buffer (pH 7.4). On addition of 20 mM sodium azide, the ESR peak height of the oleic acid-derived radicals also increased to 149 ± 3% of the control even in the 140 mM phosphate buffer under the 1.95 J/cm2 UVA irradiation (Table 1). On addition of 0.2 mM sodium azide, the ESR peak height also increased to 157 ± 27% of the control under the 1.95 J/cm2 UVA irradiation (Table 1). Thus, sodium azide seems to enhance the radical formation under various conditions. To investigate which radicals form in the standard reaction mixture with sodium azide, HPLC-ESR and HPLC-ESR-MS analyses were performed (Figs. 6 and 7). The HPLC-ESR analysis detected four peaks (peaks 1–4) at the retention times of 37.6 min, 46.5 min, 49.0 min and 51.8 min. Based on the retention times, the peaks 1 and 3 correspond to Rf radicals and 4-POBN/7-carboxyheptyl radical adducts, [M + H]+. The HPLC-ESR-MS analyses of the two new peaks (peaks 2 and 4) were performed (Fig. 7). The HPLC-ESR-MS analysis of the peak 2 compound gave ions at m/z 324 (Fig. 7A). The ions at m/z 324 correspond to the protonated molecules of the 4-POBN/6-carboxyhexyl radical adducts, [M + H]+. The HPLC-ESR-MS analysis of the peak 4 compound gave ions at m/z 352 (Fig. 7B).

Fig. 6

The HPLC-ESR analysis of the standard reaction mixture with 20 mM sodium azide. The reaction and HPLC-ESR conditions were as described in Materials and Methods except for the total volume and the composition of the reaction mixture. The total volume of the reaction mixture was 3.0 ml. The standard reaction mixture with 20 mM sodium azide was analyzed using the HPLC-ESR.

Fig. 7

HPLC-ESR-MS analyses of the peaks 2 and 4 observed for the standard reaction mixture with sodium azide. The reaction and HPLC-ESR-MS conditions were as described in Materials and Methods except for the total volume and the composition of the reaction mixture. The total volume of the reaction mixture was 3.0 ml. The standard reaction mixture with 20 mM sodium azide was analyzed using the HPLC-ESR-MS. (A) Peak 2. (B) Peak 4.

To investigate the effect of SOD on the radical formation, the ESR measurements were performed for the standard reaction mixtures with SOD (Table 1). On addition of 300 units/ml SOD under the 1.95 J/cm2 UVA irradiation, the ESR peak height kept unchanged (Table 1).

To know whether or not hydrogen peroxide is involved in the radical formation, the effect of catalase was examined. When 1260 units/ml catalase was added to the standard reaction mixtures under the 1.95 J/cm2 UVA irradiation, no change was observed in the ESR peak height (Table 1).

Discussion

In the present investigation, it was shown that the 7-carboxyheptyl radical form in the standard reaction mixture under the UVA irradiation. SOD and catalase exerted little effect on the radical formation. Superoxide anions and hydrogen peroxide do not mainly participate in the formation of the oleic acid-derived radicals. When the reaction was performed in D2O, the ESR peak height increased. Thus, singlet oxygens seem to be involved in the formation of the oleic acid-derived radicals because D2O increases the lifetime of singlet oxygens.(

A possible reaction path for the formation of the 7-carboxyheptyl radical is as follows (Fig. 8). The excited singlet state sensitizer 1(Riboflavin)*, which is produced under the UVA irradiation turns to the excited triplet state 3(Riboflavin)* through intersystem crossing. The excited triplet state 3(Riboflavin)* may reacts with triplet oxygen to form singlet oxygen (Eq. 1).(

Fig. 8

A possible reaction path for the formation of the 7-carboxyheptyl radical (Peak 3) in the standard reaction mixture.

The reaction of oleic acid with the singlet oxygen results in the formation of LOOH (9-hydroperoxy-10-octadecenoic acid or 10-hydroperoxy-8-octadecenoic acid) through the singlet oxygen ene reaction (Eq. 2).(

Ferrous ions possibly catalyze decomposition of the 9-hydroperoxy-10-octadecenoic acid. This reaction yields a radical intermediate LO•[1-(7-carboxyheptyl)-2-decenyloxyl radical]. The β-scission of the LO• results in the generation of the 7-carboxyheptyl radical. The reaction of ferrous ion with the 10-hydroperoxy-8-octadecenoic acid would result in the formation of octyl radical. We could not detect the radical. Since the 4-POBN used in this experiment is a relatively polar molecule, octyl radical which is located at the non-polar area may be difficult to be trapped by the 4-POBN.

Addition of azide to the standard reaction mixture interestingly resulted in the increase of the ESR peak height and appearance of the two new peaks (peak 2 and 4) on the elution profile of the HPLC-ESR analysis. On addition of the singlet oxygen scavenger, azide, a reactive compound which can subtract hydrogen atoms from oleic acid or Rf may form in the reaction mixture. The H-abstraction of oleic acid at C8 result in the formation of 8-hydroperoxy-9-octadecenoic acid (Fig. 9). Ferrous ions possibly catalyze decomposition of the 8-hydroperoxy-9-octadecenoic acid. This reaction yields a radical intermediate LO•[1-(6-carboxyhexyl)-2-undecenyloxyl radical]. The β-scission of the LO• could result in the generation of the 6-carboxyhexyl radical (Fig. 6, peak 2). On the other hand, The H-abstraction of oleic acid at C11 could result in the formation of heptyl radical. We could not detect the radical. Since the 4-POBN used in this experiment is a relatively polar molecule, heptyl radical which is located at the non-polar area may be difficult to be trapped by the 4-POBN. The H-abstraction of Rf could result in the formation of Rf radical.

Fig. 9

A possible reaction path for the formation of the 6-carboxyhexyl radical (Peak 2) in the standard reaction mixture with sodium azide.