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Quantification of Naturally Occurring Pyrrole Acids in Melanosomes

May 16, 2008

By Ward, Weslyn C Lamb, Erin C; Gooden, David; Chen, Xin; Burinsky, David J; Simon, John D

ABSTRACT Three naturally occurring pyrrole acids were found in Sepia, human black hair, and bovine choroid and iris melanosomes using high-performance liquid chromatography and mass spectrometry- pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA). PDCA and PTCA are common markers quantified from oxidative degradation of eumelanins. Using standards, the amounts of naturally occurring PDCA and PTCA were determined and compared to those obtained following peroxide oxidation of the same samples. Because the naturally occurring acids are water soluble, these results indicate that care must be exercised when comparing PDCA and PTCA yields from the degradation analyses of melanins isolated and prepared by different methods. This work also establishes that PTeCA is a naturally occurring pyrrole acid in melanosomes.

INTRODUCTION

Despite significant effort over the past several decades, the molecular structure of melanin remains undetermined. In the case of eumelanin (black pigment), the Raper-Mason scheme for melanogenesis indicates that the pigment is largely built from two indole derivatives, 5,6-dihyroxyindole (DHI), and 5,6-dihydroxyindole-2- carboxylic acid (DHICA) (1). To characterize and quantify eumelanin content in naturally occurring melanosomes, researchers generally perform an oxidative analysis-using either permanganate or peroxide oxidation-and the subsequent quantification of pyrrole-2,3- dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) from an originally known mass of melanosomes is then used to infer the relative contributions of DHI and DHICA to the constituent melanin pigment (2,3). Pioneering oxidation experiments identified pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) in small amounts by TLC (4-6), but later reports discredit this finding, claiming PTeCA to be exclusively an artifact of the oxidation process (7). Current oxidation experiments eliminate PTeCA (natural and artificial) altogether by using 1 M H^sub 2^SO^sub 4^ as the reaction medium (8) or by acidifying to pH 1 with 6 M HCl postoxidation (9,10). Nonetheless, oxidative analysis is a powerful technique for providing insight into the molecular composition of melanins.

The relationship between the yields of PDCA and PTCA following oxidation of the pigment and the DHI:DHICA ratio comprising the pigment assumes that all of the PDCA and PTCA measured results from pigment degradation. Herein we use high-performance liquid chromatography (HPLC) with UV absorbance at 269 nm to quantify naturally occurring pyrrole acids in Sepia, human black hair, and bovine choroid and iris melanosomes. Electrospray mass spectrometry confirmed the identification of the pyrrole acids through the appearance of appropriate parent and product ions in the mass spectra and collision-induced dissociation (CID) experiments. The naturally occurring concentrations of pyrrole acids are compared to those obtained by peroxide degradation of the same samples. In addition to these two common pyrrole acids-PDCA and PTCA-this work establishes that PTeCA is present naturally in these melanosomes.

MATERIALS AND METHODS

Ink sacs of Sepia officinalis were freshly dissected from wild cuttlefish and shipped overnight on a cold pack (Richard K. Stride, Dorset, UK). Upon arrival, melanin was extracted from the ink sac and purified using nanopure water (18.2 MOmega obtained by using a Millipore Simplicity(TM) system, Billerica, MA) according to previously published methods (11). Centrifugation was performed using an Eppendorf Centrifuge 5810R at 4[degrees]C. The purified melanin was lyophitized and stored in a desiccator for further use.

Black Indonesian human hair was purchased from R. Parrino Hair Goods (Northport, New York). Proteinase K, papain, Protease E and Triton X-100 were purchased from Sigma Aldrich (St. Louis, MO) and were used as received. Hair melanosomes were extracted enzymatically according to a previously published procedure (11).

Mature bovine eyes were ordered from Animal Technology (Tyler, TX). The eyes were collected within an hour of death and shipped overnight on ice. Details of the eye dissection and uveal melanosome purification have been published recently (12). Lyophilized choroid and iris melanosomes extracted with and without Triton X-100 were stored in a desiccator prior to use.

Twenty-five milligrams of Sepia melanin, human black hair melanosomes, and bovine choroid and iris melanosomes were each suspended in 5 mL of nanopure water and mechanically shaken in the absence of light. After 1 week, the suspension was transferred to Eppendorf tubes and centrifuged at 10 000 g for 20 min at 4[degrees]C using an Eppendorf 5810R (Westbury, NY). The pellets were collected and transferred back to the original container for resuspension. The resulting supernatants were combined and transferred to 0.1 [mu]M Ultrafree-CL low-binding PVDF membrane filter tubes (Millipore, Billerica, MA), each holding ~2.5 mL. The filter tubes were centrifuged at 10 000 g for 15 min, and the supernatants were collected. The filters were rinsed with nanopure water, and the remaining particulate matter was transferred back to the original container for resuspension in a total of 5 mL of nanopure water. The supernatants were transferred into Eppendorf tubes and dried (this requires 36-48 h) using an SPD 121P Speed Vac (Thermo Scientific, Milford, MA) at 37[degrees]C. The dried supernatant pellets were resuspended in nanopure water and combined (total volume of 1 mL) for both LC/MS and HPLC analysis. In the case of Sepia melanin, the identical process was repeated weekly for four consecutive weeks. Identical single extractions of Sepia melanin were also performed after suspending for 2 and 4 days. Compared to a week, there was no significant change in the amounts of pyrrole acids by LC/MS. We chose to suspend the samples for one week to assure that all the extractions were equilibrated.

Synthesis of PTeCA.3,4-bis-ethoxycarbonyl-2,5-dioxo-hexanedioic acid diethyl ester. Diethyl oxalate (8.9 mL, 66 mmol) was added in one portion with stirring to an ice bath-cooled solution of 21 wt % NaOEt in EtOH (22.4 mL, 60 mmol). Diethyl succinate (5.0 mL, 30 mmol) was added dropwise over 5 min and the reaction mixture was stirred at 0[degrees]C for 1 h. The cooling bath was removed and stirring was continued at room temperature for an additional 20 h. Volatiles were removed in vacua and the resulting residue was dissolved in H2O (100 mL). The solution was extracted with diethyl ether (2 x 100 mL), cooled in an ice bath, and acidified to pH 1- 2 by careful addition of 4 N HCl (12 mL). The acidic solution was extracted with ethyl acetate (2 x 100 mL). The combined organic extracts were washed with saturated aqueous NaCl (50 mL) and dried (Na^sub 2^SO^sub 4^), The drying agent was removed by nitration and the filtrate was concentrated under reduced pressure to give a brown oil. Flash column chromalography on silica gel (ethyl acetate:hexanes [1:4 [arrow right] 1:1]) afforded the title compound as a colorless oil (5.5 g, 50% yield). ^sup 1^H NMR (300 MHz, CDCl^sub 3^): delta 12.7 (s, 1H), 4.59 (dd, J = 5.1 Hz, 9.0 Hz, 1H), 4.28 (m, 4H), 4.11 (m, 4H), 3.57 (s, 1H), 2.90 (m, 2H), 1.32 (m, 6H), 1.18 (m, 6H). ^sup 13^C NMR (75 MHz, CDCl^sub 3^): delta 188.5, 172.9, 170.8, 167.9, 160.4, 159.2, 102.8, 63.0, 62.2, 61.4, 49.5, 32.1, 31.5, 14.2, 14.0. MS (ESI): m/z 375 [M + H]^sup +^.

Tetraethyl pyrrole-2,3,4,5-tetracarboxylic acid. A mixture of 3,4- bisethoxycarbonyl-2,5-dioxo-hexanedioic acid diethyl ester (1.6 g, 4.3 mmol) and ammonium carbonate (1.5 g, 16 mmol) in glacial acetic acid (6 mL) was heated at 90[degrees]C (oil bath temperature) for 4 h. Upon cooling to room temperature, the reaction mixture was made basic (pH 8-9) by the addition of saturated aqueous NaHCO^sub 3^ and extracted with ethyl acetate (3 x 50 mL). The combined organic extracts were washed with H2O (50 mL), saturated aqueous NaCl (50 mL) and dried (Na^sub 2^SO^sub 4^). The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. Purification of the resulting brown oily residue by flash column chromatography on silica gel (ethyl acetate:hexanes [1:7 [arrow right] 1:1]) afforded the title compound as a clear, yellow oil (200 mg, 13% yield). ^sup 1^H NMR (300 MHz, CDCl^sub 3^): delta 10.2 (bs, 1H), 4.32 (m, 8H), 1.32 (m, 12H). ^sup 13^C NMR (75 MHz, CDCl^sub 3^): delta 163.1, 159.1, 123.7, 122.0, 62.2, 61.7, 14.3, 14.2. MS (ESI): m/z 356 [M + H]^sup +^ , m/z 378 [M + Na]^sup +^.

pyrrole-2,3,4,5-tetracarboxylic acid (1). Powdered KOH (0.338, 5.12 mmol) was added in one portion at room temperature to a solution of tetraethyl 2,3,4,5-pyrroletetracarboxylate (186 mg, 0.523 mmol) in ethanol (3.2 mL) and the reaction mixture was refluxed for 12 h. Volatiles were removed in vacuo and the residue was dissolved in H2O (3.2 mL). The solution was cooled in an ice water bath and was acidified to pH 0-1 by dropwise addition of 12 M HCl. The resulting precipitate was removed by vacuum filtration, washed with H2O (3 mL) and dried in vacuo to give the desired acid as a white solid (86 mg, 68% yield). ^sup 1^H NMR (300 MHz, DMSO- d^sub 6^): delta 12.0 (bs, 1H). ^sup 13^C NMR (75 MHz, DMSO-d^sub 6^): delta 169.5, 160.4, 130.4, 119.6. MS (ESI): m/z 242 [M - H]^sup -^. Quantification of pyrrole acids using HPLC-UV 269 nm. Standards of PTCA and PDCA were diluted to a concentration of 20 ng [mu]L^sup – 1^ in nanopure water. Injections ranging in volume from 0.5 to 10 [mu]L of the standards (each separately) were introduced into an Agilent 1100 HPLC system witha C18 column (Zorbax 2.1 x 150 mm, 5 [mu]m) and a diode array detector. The detector was programmed to collect the full UV absorption spectrum and to display the absorbance at 254, 269, 280 and 330 nm using a bandwidth of 16 nm and reference wavelength of 600 nm (50 nm bandwidth). The isocratic mobile phase, consisting of 99% 0.1 M potassium phosphate adjusted to pH 2.1 and 1% methanol, was pumped at a flow rate of 0.2 mL min^sup -1^. Chemstation software (Agilent Technologies, Santa Clara, CA) was used to integrate the A^sub 269^ peak area for serial injections of the PTCA and PDCA standards (10-200 ng). Standard curves for PTCA and PDCA were prepared by extrapolating the line of best fit with R^sup 2^ values of at least 0.99 for each compound.

To quantify the amount of PTCA and PDCA in each eumelanin- containing sample, serial injections ranging from 1 to 20 [mu]L of the supernatant were subjected to the above HPLC conditions. Linear regression analysis of the PTCA and PDCA standards was used to determine the ng (PTCA or PDCA) per mg of eumelanin (corrected to initial dry weight). The final ng PTCA per mg of Sepia melanin was determined by adding the ng mg^sup -1^ collected from each of the four consecutive weeks. An identical analysis was performed using an absorbance of 254 nm. The ratio of A^sub 269^/A^sub 254^ for total PTCA was 0.95, and the A^sub 269^/A^sub 254^ for PDCA was 1.42.

A compound of interest eluted approximately 4.5 min after PTCA, yielding a UV absorption maximum at 282 nm and a deprotonated molecule ([M - H]^sup -^, m/z 242) in its mass spectrum under negative ESI conditions. The HPLC retention time, UV absorbance and molecular mass of this unknown compound matched those of a synthesized standard of pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA).

Mass spectrometry. The ESI source was operated in the negative ion mode under the following conditions: spray voltage, 3.5 kV; skimmer voltage, -25 V; capillary exit voltage, -100 V; nitrogen drying gas temperature and flow rate, 350[degrees]C and 9 L min^sup – 1^, respectively; nebulizer gas pressure, 45 psi; fragmentation amplitude ramped from 30% to 200% of 1.0 V. Data-dependent MS/MS (Auto MS/MS) scans were performed using Agilent LC/MSD Trap 5.3 version software, by which the mass spectrometer acquired an ESI mass spectrum and determined the individual ions in the spectrum. The instrument then switched to MS/MS operation and MS/MS product ion spectra were acquired for each ion in the original mass spectrum, provided that these ions fulfilled certain criteria. All data acquired were processed by Agilent Chemstation Rev. A. 09.01 software (Agilent, Palo Alto, CA).

Identification of pyrrole acids using HPLC/MS. The resuspended supernatants of Sepia melanin (in water) were injected onto a C18 column (Zorbax 2.1 x 50 mm, 3.5 [mu]m) and eluted using a 10 min linear gradient, from 0% to 100% in component B. The mobile phase components were A = 95:5 vol/vol H^sub 2^O/CH^sub 3^CN, and B = 95:5 vol/vol CH^sub 3^CN/H^sub 2^O. Although a variety of conditions were attempted, adequate separation was not readily achieved. However, using the specificity of tandem mass spectrometry to compensate for insufficient chromatographic separation, it was possible to readily identify PDCA and PTCA, along with PTeCA, each ionizing in the negative ESI mode affording [M - H]^sup -^ ions at m/z 154, 198 and 242, respectively.

RESULTS

Figure 1 shows the HPLC/UV trace (269 nm) using the conditions described previously. The peaks corresponding to PDCA, PTCA and PTeCA are indicated in the figure. PDCA and PTCA were identified using standards provided by Shosuke Ito, while the PTeCA standard was synthesized as described in the literature. The MS and HPLC figures are from Sepia melanin; PDCA and PTCA for the hair and ocular melanosomes show the same fragmentation information and approximate retention times as Sepia. The chromatogram of the aqueous extract from hair melanosomes (Fig. 2) contains an additional major peak, identified as PTeCA. Slight variations in the mobile phase pH and composition caused retention time shifts for the hair sample (analyzed 9 months after the Sepia sample), so new calibration curves were prepared for each sample analysis.

Figure 1. HPLC chromatogram (269 nm) of the water-solubilized constituents from Sepia melanosomes. The peaks corresponding to naturally occurring pyrrole acids are indicated.

Figure 3 shows the mass spectrum for the aqueous extract of Sepia eumelanin. We focus on the peaks labeled in bold at m/z 110.1, 153.9, 197.8 and 241.8. The m/z 110.1 peak corresponds to the [M - H]^sup -^ ion for mono-pyrrole acid. Figure 4 shows the CID product ion spectrum of the [M - H]^sup -^ ion (m/z 198) for (A) the PTCA standard, and (B) the aqueous supernatant of Sepia melanin. The identical fragmentation patterns confirm that the naturally occurring species is PTCA. Scheme 1 illustrates the fragmentation pathway for PTCA. A similar comparison of the CID product ion spectrum for [M - H]^sup -^ at m/z 153.9 with that obtained for a PDCA standard confirms that this compound also occurs naturally. The HPLC/UV (269 nm, A^sub 600^) chromatogram of PTCA standard, shown in Fig. 5A, demonstrates that the PTCA standard is reasonably pure. However, the mass spectrum shown in Fig. 5B appears to contain signals belonging to both PDCA and pyrrole-CA. This apparent confusion stems from a facile in-source decarboxylation (elimination of CO2) of the deprotonated molecule ([M - H]^sup -^) at m/z 198, producing the signals at m/z 154 and 110. Because of the unfortunate appearance of these coincident signals (i.e. ions that may represent either fragmentation products or intact molecular species from homologous compounds), mass spectrometric peak areas and/or precursor-to-product ion transitions should not be used to quantify PTCA without using internal standardization, and paying scrupulous attention to tuning conditions, which may exacerbate the in-source fragmentation reactions, and other instrument-dependent variations (13).

Figure 2. HPLC chromatogram (269 nm) of the water-solubilized constituents from human black hair melanosomes. The peak at 20.87 min corresponds to pyrrole-2,3,4,5-tetracarboxylic acid.

Figure 3. Mass spectrum of the water-solubilized constituents from Sepia melanosomes.

Figure 4. Collision-induced dissociation product ion spectrum of the m/z 198 ion ([M - H]^sup -^) from (A) pyrrole-2,3,5- tricarboxylic acid (PTCA) standard, and (B) the water-solubilized constituents of Sepia melanin. Identical fragmentation patterns confirm that the naturally occurring species is PTCA. Scheme 1 illustrates the fragmentation pathway followed by PTCA.

Scheme 1. Fragmentation pathway showing the most abundant product ions of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3- dicarboxylic acid (PDCA). Loss of CO2 from the carboxylic acid substituents dominates the dissociation pathways due to the thermodynamic stability of the pyrrole ring.

Figure 5. HPLC/UV (269 nm, A^sub 600^) chromatogram of pyrrole- 2,3,5-tricarboxylic acid (PTCA) standard (A) indicating the relative purity of the PTCA standard. However, the mass spectrum shown (B) appears to contain pyrrole-2,3-dicarboxylic acid, along with the pyrrole-CA. These coincidental ions (m/z 154 and 110) are artifacts of the analysis, resulting from fragmentation of the PTCA anion under some tuning conditions.

Figure 6. Collision-induced dissociation product ion spectrum of m/z 242 from the Sepia sample. The mass and UV absorbance behavior (269 nm, A^sub 600^) of the naturally occurring material are consistent with those of synthetic pyrrole-2,3,4,5-tetracarboxylic acid, thus confirming its identity.

Table 1. Quantification (ng mg^sup -1^) of PDCA, PTCA and PTeCA from the aqueous extracts of melanosomes. The corresponding yields of PDCA and PTCA from hydrogen peroxide oxidation are also listed.

Table 2. Yield of pyrrole acid (ng mg^sup -1^) for successive aqueous extractions of naturally occurring Sepia eumelanin.

The predicted structure and CID product ion spectrum of the m/z 242 ion are shown in Fig. 6. The [M - H]^sup -^ and HPLC-UV (269 nm, A^sub 600^) absorbance of synthetic PTeCA confirmed this assignment. UV detection of PTeCA eliminated the possibility that PTeCA was created by artifact-no harsh conditions or external voltages were applied, indicating that its existence is natural. The HPLC-UV (269 nm, A^sub 600^) of the aqueous extract from human hair melanosomes contains a substantially greater peak area for PTeCA (20.9 min) as shown in Fig. 2.

Table 1 summarizes the amount of pyrrole acids naturally occurring in aqueous eumelanin extracted from Sepia, black hair (2% Triton X-100 extract), and bovine choroid and iris melanosomes (with and without 1% Triton X-100). Sepia was isolated without the use of Triton X-100. In addition, our previously published data obtained from these same samples for the yield of PDCA and PTCA from H^sub 2^O^sub 2^ degradation is presented. Table 2 quantifies the yields of the PDCA and PTCA (ng mg^sup -1^) for successive aqueous extractions of naturally occurring Sepia eumelanin. These comparisons show that the amount of naturally occurring pyrrole acid is a considerable portion (24% for PDCA, 22% for PTCA) of that found following degradation analysis of the sample.

DISCUSSION

Information about the composition of melanin is usually obtained from marker compounds formed by oxidative degradation (2,3). This study presents the first observation of naturally occurring PDCA, PTCA and PTeCA, from melanosomes. Oxidative degradation protocols typically terminate with acid addition to eliminate any artifactual formation of PTeCA (8-10). Unfortunately, naturally occurring PTeCA would then also be hydrolyzed, most likely to PTCA. This transformation is one of the factors that can potentially skew the reliability of oxidative degradation analyses and/or comparisons. Factors such as the presence of naturally occurring pyrrole acids, number of washes employed during melanosome isolation and use of surfactant may also cause a misrepresentation of DHI and DHICA content. Quantifying and comparing the naturally occurring pyrrole acids in aqueous melanosome extracts minimizes the likelihood of a false interpretation of the pigment composition. Additionally, these results establish that HPLC separation with UV absorbance measurement is the preferred method of quantification, eliminating the possibility of CO2 loss, a process that can complicate the interpretation of mass spectrometric results. Quantitative studies of PTCA and PDCA using either peroxide or permanganate oxidation assume that negligible amounts of pyrrole acids exist in melanosomes prior to oxidation. Intentional oxidation is believed to be the sole source of PDCA and PTCA, derived from DHI and DHICA, respectively. However, our findings show the acids to exist naturally, and the propensity of PTCA to naturally decompose is dependent upon the melanosome. This observation raises the question of how much of the pyrrole acid is lost during isolation.

Triton X-100 is the most commonly used nonionic surfactant for solubilizing membrane proteins during the isolation of tissue- embedded melanosomes. Polyoxyethylene-type surfactants are susceptible to peroxide formation during production, storage or use with peroxide formation being enhanced by exposure to light or air (14). Any peroxides formed during the Triton X-100 centrifugation and/or washing of the samples may cause premature oxidation of the melanosomes, forming PTCA (that is lost during this step). The DHI, and subsequently PDCA, do not appear to be affected by Triton X- 100. The ratio of DHI:DHICA is often used to characterize melanosomes. As DHICA, but not DHI, is affected by Triton X-100, and Triton X-100 affects some melanosomes more than others, comparing DHI:DHICA for dissimilar samples may not be a sound strategy.

The addition of Triton X-100 during melanosome isolation has a more profound impact on iris than choroid, releasing 3.3 and 1.5 times more naturally occurring PTCA with Triton X-100 than without, respectively. Infrared spectroscopy indicates that iris melanosomes have a higher degree of conjugation than choroid melanosomes (15). By facilitating oxidation, Triton X-100 may affect this conjugation in iris, releasing more PTCA. Whereas, choroid melanosomes, with a less conjugated network, are not as heavily affected by the addition of Triton X-100.

Previous reports indicate that PTeCA is an artifact of the oxidative degradation process (7). Acidifying the product of oxidative degradation eliminates variability of the artifact amounts. Our findings show that the PTeCA content varies between melanosome samples-the ratio of PTeCA:PTCA for hair melanin is ~23 times higher than that of Sepia melanin (based on Figs. 1 and 2). Very small amounts of PTeCA were present in ocular melanosomes, as determined by comparing the peak area for equivalent injection amounts of Sepia, hair and ocular aqueous melanin extractions. Transforming all PTeCA postoxidative degradation of hair melanin could potentially raise the PTCA content roughly by 15-20%. However, because PTeCA is also a byproduct of the oxidation process, its removal is unavoidable if only PTCA is assayed to determine pigment composition.

In our previously published studies on the binding of melanin to Sepia, we noted that H^sub 2^O^sub 2^/OH- oxidation of EDTA-washed samples gave a lower yield of PTCA (12 130 ng mg^sup -1^ vs 16 600 ng mg^sup -1^ before EDTA washing) (16). We had concluded from these measurements that repeated soaking in EDTA solutions and repeated water washing to remove extra EDTA meant that oligomers rich in DHICA are solubilized in aqueous solution once the doubly charged metal ions are removed by the EDTA (16). It is interesting to rethink this conclusion based on the current measurements. The data in Table 1 indicate that the EDTA treatment and subsequent washing of Sepia eumelanin will also serve to remove naturally occurring PDCA and PTCA. The difference in PTCA yield between the natural and EDTA-treated samples is 16 600 – 12 130 = 4470 ng mg^sup -1^. Four water extractions of Sepia sample dissolve a total of 3700 ng mg^sup -1^ PTCA from the sample. Thus, at least 83% (3700/4470) of the decreased yield of PTCA from the degradation for the EDTA sample arises from the dissolution of naturally occurring PTCA, not a solubilization of DHICA-rich oligomers, as originally thought.

The origin of the naturally occurring pyrrole acids is not known. They could result from oxidation of the pigment under natural conditions, or may be formed as necessary constituents or byproducts of melanogenesis. What is clear is that the experimental approach used herein did not inadvertently generate these acids. If successive washings had generated these species by oxidation, the amounts of pyrrole acids for each extraction would be the same. The data in Table 2 clearly show that this is not the case and that there is an equilibrium between melanosome-bound and dissolved acids. These results may also be relevant to a recent study suggesting that the concentration of PTCA in human hair melanosomes may be a better risk indicator for melanoma than the traditional classifications of skin phenotypes (17).

Acknowledgements-We thank Shosuke Ito and Kasumasa Wakamatsu for providing PDCA and PTCA standards. This work was supported by the Medical Free Electron Laser Program and Duke University.

REFERENCES

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Weslyn C. Ward1, Erin C. Lamb1, David Gooden1, Xin Chen1, David J. Burinsky2 and John D. Simon*1

1 Department of Chemistry, Duke University, Durham, NC

2 GlaxoSmithKline, Research Triangle Park, NC

Received 8 November 2007, accepted 4 January 2008, DOI: 10.1111/ j.1751-1097.2008.00328.x

[dagger] This invited paper is part of the Symposium-in-Print: Melanins.

* Corresponding author email: john.simon@duke.edu (John D. Simon)

(c) 2008 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/08

Copyright American Society for Photobiology May/Jun 2008

(c) 2008 Photochemistry and Photobiology. Provided by ProQuest Information and Learning. All rights Reserved.




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