Ultraviolet A Induces Generation of Squalene Monohydroperoxide Isomers in Human Sebum and Skin Surface Lipids In Vitro and In Vivo  Swarna Ekanayake Mudiyanselage, Peter Elsner, Jens J. Thiele  Journal of Investigative Dermatology  Volume 120, Issue 6, Pages 915-922 (June 2003) DOI: 10.1046/j.1523-1747.2003.12233.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Human sebum squalene is dose dependently depleted by UVA (B), but not by UVB (A). Sebum was collected from healthy volunteers using Sebutapes®, irradiated and immediately subjected to lipid extraction. Subsequently, squalene levels were measured by HPLC using UV detection as described. Shown are means±SEM, (A) n=7, (B) n=10. *p<0.05, **p<0.01; ***p<0.001 versus untreated control; n.s.=not significant. Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Identification of USLPP as squalene monohydroperoxides by HPLC and LC-MS analysis. Chromatograms of UVA irradiated human sebum (80 J/cm2 UVA) and of purified synthetic SqmOOH recorded by UV detection at 210nm show the same retention time (A). APCI mass spectrum of the peak at 4.50 min of the irradiated sebum, corresponding to squalene monohydroperoxides (B). APCI mass spectrum of the squalene peak at 9.50 min (C). Conditions: Hypersil ODS column (5 μm; 250 x 4 mm i.d.); methanol:ethanol (1:1, v/v); detection at 210 nm; 1.2 ml/min. SQ is an unsaturated hydrocarbon which ionized very poorly. The hydroperoxy moiety in SqmOOH confers better ionization, but the molecule becomes more labile. Therefore, two intense (and diagnostic) fragment ions (m/z 409 and 425) occur together with a weak molecular ion at m/z 443. The mass spectra do not reflect the relative amounts. Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 A. Structure of squalene. B. 1H NMR spectral data of SqmOOH purified from sebum. Partial structures (A-C) encountered for the hemiterpene units in SqmOOH, and the corresponding 1H NMR chemical shifts are shown. Assignments are based on comparison with 1D and 2D data measured for a squalene reference, and on a 1D 1H spectrum and a 2D adiabatic TOCSY (WURSTm) experiment with the SqmOOH Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Correlation of decrease of squalene and increase of SqmOOHs in human sebum after UVA irradiation. Sebum was collected from healthy volunteers using Sebutapes_, irradiated and immediately subjected to lipid extraction. Levels of squalene and SqmOOH were measured simultaneously by HPLC using UV detection as described. Shown are means±SEM, n=10; correlation coefficient r=-0.97; p< 0.005. (□ SqmOOH; ▴ SQ) Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Dose dependent increase of squalene monohydroperoxides (SqmOOH) in human sebum immediately after irradiation with UVB (A) or UVA (B). Sebum was collected from healthy volunteers using Sebutapes_, irradiated and immediately subjected to lipid extraction. Subsequently, SqmOOH levels were measured by HPLC using UV detection as described. Shown are means _ SEM, (A) n=7, (B) n=10, **p<0.01, ***p<0.001 versus untreated controls. Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Squalene monohydroperoxides are generated in skin surface lipids upon UVA exposure of human skin in vivo. Formation of SqmOOH was compared upon exposure of either sebum (in sebutapes; in vitro) or human skin (foreheads of volunteers; in vivo) to single doses of UVB and UVA (60 mJ/cm2 UVB, and 20 J/cm2 UVA, respectively). After irradiation, ethanol extracts from Sebutapes (n=10; sebum) or human forehead skin (n=4; skin surface lipids) were analyzed for SqmOOH content as described. Shown are means±SD. Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 HPTLC analysis confirming generation of specific squalene monohydroperoxide isomers in UVA irradiated human sebum. Visualization of analytes with Godin's reagent (A); with N,N-DPDD reagent (B). The following samples were separated: Lane 1: sebum taken without UV irradiation; 2: sebum taken after irradiation with 80 J/cm2 UVA; 3: SqmOOH, isolated by collection of HPLC fraction from irradiated sebum; 4: synthetic SqmOOH; 5: squalene. The amount of sebum separated in lanes 1 and 2 is 60 μg. SQ: squalene; SE: sterol esters; FA: fatty acids; PL: polar lipids; X: SqmOOH isomers. Journal of Investigative Dermatology 2003 120, 915-922DOI: (10.1046/j.1523-1747.2003.12233.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions