RP and sodium azide (NaN₃) were purchased from the Sigma Chemical Co. (St. Louis, MO). All other reagents were obtained through commercial sources and were the highest quality available. All solvents used were HPLC grade.

The UVB and UVA light boxes were custom made 4-lamp units with 2ft fluorescent lamps spaced approximately 7 mm apart. The emissions from each source were unfiltered, and the high output UVB and UVA lamps were obtained from National Biologics (Twinsburg, OH). The irradiance of the UVB and UVA light sources was determined using an Optronics OL-754 Spectroradiometer (Optronics Laboratories, Orlando, FL), and the light dose was routinely checked using a Solar Light (Philadelphia, PA) PMA-2200 radiometer equipped with either a Solar Light PMA-2110 UVA detector or Solar Light PMA-2101 UVB detector.

As shown in Figure 2, the maximum emission of the high output UVA fluorescent lamps was 351–352 nm, with a spectral distribution (left spectrum in Figure 2) as follows: UVC (250–280 nm), 7.80 × 10⁻¹⁰ Watts/cm²; UVB (280–315 nm), 4.03 × 10⁻⁶ Watts/cm²; UVA (315–400 nm), 3.68 × 10⁻⁴ Watts/cm². Thus, the distribution of ultraviolet radiation in the UVA source was 0.0002% UVC, 1.1% UVB, and 98.9% UVA.

The maximum emission of the unfiltered high output UVB fluorescent lamps was 313 nm, with a spectral distribution (right spectrum in Figure 2) as follows: UVC (250–280 nm), 1.01 × 10⁻⁵ Watts/cm²; UVB (280–315 nm), 1.16 × 10⁻³ Watts/cm²; UVA (315–400 nm), 9.95 × 10⁻⁴ Watts/cm². Thus, the distribution of ultraviolet radiation was 0.5% UVC, 53.6% UVB, and 46.0% UVA.

A solution (2–3 mL) of 0.5 % RP dissolved in ethanol was placed in a UV-transparent cuvette and photoirradiated under the high output UVB lamps for a period of time required to deliver a UVB light dose of 7, 14, or 21 J/cm². The reaction mixture was then concentrated to about 200 μL under reduced pressure. Reversed-phase HPLC separation of the resulting photodecomposition products was accomplished using a Prodigy 5 μ ODS column (4.6 × 250 mm, Phenomenex, Torrance, CA) eluted isocratically with 10% methylene chloride in methanol (v/v) at 1 mL/min.

Samples containing 100 mM methyl linoleate and 1.0 mM RP in ethanol were placed in a UV-transparent cuvette and irradiated under the high output UVB lamps for a period of time required to deliver UVB dose of 0, 7, 14, 21, or 35 J/cm² of UVB light for the first set of experiments and with 0, 2.5, 5.0, 7.5, or 10 J/cm² of UVB light for the second set of experiments. The levels of lipid peroxidation were measured by quantification of the methyl linoleate hydroperoxides from the HPLC peak area at 235 nm [7, 8, 12]. Methyl linoleate hydroperoxides were separated by HPLC using a Prodigy 5 μ ODS column (46 × 250 mm, Phenomenex, Torrance, CA) stationary phase and 10% water in methanol (v/v) as the mobile phase at 1 mL/min [7, 8].

The effect of 20 mM NaN₃, a singlet oxygen and free radical scavenger, on the levels of lipid hydroperoxide formation induced by photoirradiation of RP with UVB light was investigated using irradiation conditions described above.

NaN₃ is a singlet oxygen and free radical scavenger. To further confirm the formation of singlet oxygen, one of the ROS species, involved in the lipid peroxidation, photoirradiation of RP with UVB light was determined in the presence of H₂O or deuterium oxide (D₂O) (enhancement of singlet oxygen lifetime) under experimental conditions as described above. The amount of deuterium oxide and water was 20% in methanol.

Statistical comparison between experimental groups was performed with the General Linear Model (GLM) using the SAS software, version 8.0 (SAS Institute Inc., Cary, NC). Statistically significant differences were identified when p ≤ 0.05.

A Waters HPLC system consisting of a Model 600 controller, a Model 996 photodiode array detector, and a pump was used for the separation and purification of photodecomposition products of RP.

Photoirradiation of RP in ethanol with 7, 14, or 21 J/cm² UVB light was conducted and the photodecomposition products were separated by reversed phase HPLC. The HPLC profile of RP irradiated with 14 J/cm² UVB light is shown in Figure 3 (Panel B). For structural identification of the photodecomposition products, photoirradiation of RP with UVA light under similar conditions as previously reported (7) was performed in parallel and the resulting HPLC profile is shown in Figure 3 (Panel A).

As such, the profiles of the photodecomposition products formed from photoirradiation of RP with the high output UVB light source and the high output UVA light source can be compared. Based on comparison of the HPLC retention time, UV-absorption spectrum and mass spectrum with those of RP, the material contained in the chromatographic peak that eluted at 26.5 min in Figure 3B was identified as the recovered substrate, RP. The material eluting at 12.7 min in Figure 3 following irradiation of RP with the high output UVB light source was 4-keto-RP as evidenced by the HPLC retention time (Figure 3), UV-visible absorption spectrum (with maximal absorptions at 335, 349, and 369 nm, respectively) (Figure 4), and mass spectrum (molecular ion at m/z 540) (data not shown). These results are identical to those of 4-keto-RP formed from photoirradiation of RP with the high output UVA light source (Figure 3A) and formation of 4-keto-RP prepared synthetically by oxidation of RP with m-chloroperbenzoic acid [7].

Following irradiation of RP with the high output UVB light, the compounds that eluted at 9.8 and 10.2 min (Figure 3) each contained more than one compound, which is similar to the results previously reported for the photoirradiation of RP with UVA light [7]. Mass spectral analysis indicated that each peak contained molecular ions at m/z 586 and characteristic fragment ions at m/z 571 (loss of a methyl group), m/z 540 (loss of a molecule of ethanol), m/z 287 and 247 (data not shown). By comparison of their HPLC retention times (Figure 3A and B), mass spectra, and UV absorption spectra with the standards characterized previously from photoirradiation of RP with UVA light [7], these photodecomposition products were identified as 11-ethoxy-12-hydroxy-RP and 13-ethoxy-14-hydroxy-RP, with each peak containing a mixture of the trans- and cis isomers [7].

The material contained in the chromatographic peak eluted at 7.8 min (Figure 3) following irradiation of RP with ultraviolet light had a molecular ion at m/z 268 (data not shown). The mass spectral profile, UV-visible absorption spectrum and HPLC retention time are identical to those of the synthetic standard anhydroretinol (AR) prepared from reaction of RP with hydrochloric acid [7]. Upon further separation by HPLC using a Vydac C₁₈ column, this AR was determined to be a mixture of all-trans-AR, 6Z-cis-AR, 8Z-cis-AR, and 12Z-cis-AR, with the 12Z-AR isomer as the predominant product (Figure 5).

The material contained in the chromatographic peak eluted at 6.8 min in Figure 3 had a mass spectrum with a molecular ion at m/z 314 (data not shown) which is consistent with ethoxy derivatives of AR [7]. The peak was collected and separated using a different HPLC system (Vydac C₁₈ column; isocratic 14% water in methanol mobile phase; data not shown) into four major chromatographic peaks which had nearly identical UV-visible absorption spectra (data not shown) and identical molecular ions at m/z, 314. These products are isomeric 15-ethoxy-ARs, with each tentatively assigned as 8Z-15-ethoxy-AR, 12Z-15-ethoxy-AR, 6Z-15-ethoxy-AR, and all-trans-15-ethoxy-AR, respectively. These results suggest that these compounds are formed from addition of one molecule of ethanol to AR.

The products formed when RP was irradiated with 7 and 21 J/cm² UVB light were similarly analyzed. The same photoirradiation products were identified, with differences only in the yields of each photodecomposition product (data not shown).

Photoirradiation of RP in the presence of methyl linoleate with UVB light of different light doses (0, 7, 14, 21, 28, 35, and 70 J/cm²) was conducted. Photoirradiation of RP and photoirradiation of methyl linoleate alone were also performed in parallel. As previously described, we have determined the extent of lipid peroxide formation by quantification of the amount of methyl linoleate hydroperoxides using HPLC methods [7, 12]. Photoirradiation of RP with UVB light in the absence of methyl linoleate did not produce any methyl linoleate hydroperoxides. Photoirradiation of methyl linoleate with 7.5 and 10 J/cm² UVB light produced measurable methyl linoleate hydroperoxides (Figure 6). This differs from our previous studies where irradiation of methyl linoleate with up to 35 J/cm² of UVA did not produce methyl linoleate hydroperoxides (7). Nonetheless, inclusion of RP in the methyl linoleate solution undergoing UVB irradiation resulted in increased methyl linoleate hydroperoxide formation (Figure 6). The level of lipid peroxidation induction increased linearly with the light dose up to 10 J/cm² and is significantly higher (p ≤ 0.05) than that induced by irradiating methyl linoleate in the absence of RP.

Similar to our previous study of photoirradiation of RP with UVA light [7], the involvement of free radical intermediates in the peroxidation of methyl linoleate initiated by photoirradiation of RP with UVB light was examined by adding the free radical scavenger NaN₃. As shown in Figure 6, NaN₃ significantly inhibited lipid peroxidation initiated by photoexcited RP (p ≤ 0.05).

Our results indicated that lipid peroxidation induced by photoirradiation of RP with UVB was increased by 12–15% when H₂O was replaced by D₂O (p ≤ 0.05) (data not shown). In addition to being a free radical scavenger, NaN₃ also effectively reacts with singlet oxygen (¹O₂) and hydroxyl radical [7] preventing their reaction with other molecules (e.g. methyl lineolate). Therefore, NaN₃ alone cannot be relied upon to determine whether singlet oxygen or free radicals are involved in peroxidation. Since singlet oxygen has a longer half-life in deuterium water (D₂O), use of both NaN₃ and D₂O should provide a reliable approach for determining whether singlet oxygen is involved in peroxidation. Thus, our results from the inhibition of lipid peroxidation by NaN₃ and the enhancement by D₂O indicate that singlet oxygen, formed from photoirradiation of RP with UVB light, is involved in the induction of lipid peroxidation.