Demographics and acne grading
Demographics and distribution of the counted lesions of acne patients are detailed in the Supplementary Table S1. The mean age (years) of the acne patients and of controls was 17.2 ±1.76, and 17.5±1.76, respectively. Among the affected group, 15 (50%) had mild acne while 10 (33.3%) and 5 (16.7%) had moderate and severe acne respectively.
Determination of elemental components of facial sebum
Forty-one FFAs, and 7 FOHs were quantified by GCMS (Supplementary Table S2). FFAs consisted of saturated and unsaturated species. Among saturated FFAs, several branched FFAs with one methyl branching at the iso (ibrFA) and anteiso (abrFA) positions were chromatographically resolved from the corresponding straight chain isomer with the same length. The latter ones were categorized as even (eFA) and odd (oFA) FFAs according to their even or odd number of C-atoms. The ibrFAs, and abrFAs subgroups included 7 members each, with C-atoms ranging from 12 to 18. The subclasses of eFAs, oFAs, MUFAs, and PUFAs included 8, 7, 10, and 2 members, respectively. The eFAs and oFAs together were SFAs with chain length ranging from 12 to 26 carbon atoms. MUFAs had mainly an even C-number, with the exception of C15:1, and C17:1. The latter one was found in both straight and branched isomers, which were assigned tentatively according to GCMS criteria. The C16:1 MUFA was characterized as sapienate (C16:1n-10) based on the same elution time and MS spectrum of the authentic reference compound and the resolution from palmitoleate (C16:1n-7), which eluted later. Palmitoleate was excluded from the quantitation due to its negligible amounts in sebum. The C18:1 MUFA appeared as a single peak. Likely, C18:1 isomers were not separated under the used GCMS conditions. FOHs with a chain length between 14 and 26 carbon atoms, were either confirmed with authentic compounds or tentatively assigned based on their MS spectrum and elution time (Supplementary Table S2). To visualize the within-class distributions of FFAs and FOHs, their amounts on foreheads and cheeks were averaged and plotted in radar diagrams (Supplementary Figure S3). Polarization of FFAs and FOHs distribution was apparent in both NA and A groups. The top 3 members, ordered according to their abundance, were, in each subclass, as follows: 13Me-C15:0 > 14Me-C16:0 > 12Me-C14:0 for the ibrFAs; 12Me-C15:0 > 15Me-C18:0 > 13Me-C16:0 for the abrFAs; C16:0 > C18:0 >> C14:0 for eFAs; C15:0 > C17:0 > C21:0 for the oFAs; C18:1 > C16:1 > C17:1 for the MUFAs, and FOH18:0 >> FOH16:0 > FOHC22:0 for the FOHs. The PUFA C18:2 was more abundant than C20:2.
Separation of sebum lipids by HPTLC developed bands due to six major components, cholesterol, FFAs, TGs, WEs, CEs, and squalene (Supplementary Figure S1), according to their elution order. Heat maps of the average amounts of sebum lipids in foreheads and cheeks in NA and A groups are reported in the Figure 1A and 1B, respectively. It is apparent that the amounts of most sebum lipids were significantly elevated in the A group. Inspection of the abundance profiles revealed that specific FFAs and FOHs members were affected more than others in the two sites. The supplementary Table S3 reports the amounts of sebum lipids (average µg ± SD) represented in the heat maps.
Sebum excretion rates and correlation of sebum lipids with acne lesions
Sebum weights were obtained by summing the amounts (µg) of individual lipids quantified by GCMS and HPTLC and were used to obtain the sebum excretion rates (SER), which expresses the µg secreted on surface unit per minute (µg/cm2/min). SER values were significantly higher in acne patients (supplementary Figure S2) at both face sites, as shown by the p-values in the table included. SER values determined from cheeks were higher than those obtained from foreheads in the affected group only (p<0,01). The associations of average composition of sebum from foreheads and cheeks with lesion counts was investigated by Spearman’s correlation. The amounts of squalene were positively correlated with the number of comedones (R=0.47, p< 0,05, data not shown).
ASCA analysis on the relative concentrations of individual lipids.
ASCA analysis, which is described in Appendix 1, was performed on the data set after normalization of the absolute individual amounts by the total sebum quantity (weight/weight percentage). The results of SCA analysis for the effect of the acne condition on the relative amounts on cheeks and foreheads are reported in Figure 2. The percentage of two aibrFFAs, i.e. 9Me-C12:0, and 11Me-C14:0, was lower on the forehead; whereas the ibrFFAs 10Me-C12:0, and 16Me-C18:0 had lower percentage on foreheads and cheeks, respectively. SFAs, and several MUFAs showed significantly higher percentage in the A group limited to the forehead. Percentages of short and long chain SFAs, PUFAs, and cholesterol, were decreased in A at the cheeks. Several FOHs were present at lower percentage in A, on both sites. Percentages of squalene and cholesterol were, respectively, higher and lower in the A group regardless the face site. Among the A subjects, outliers accounted for the 20 % and 27 % of the entire A group when scores were examined in the foreheads and the cheek lipid profiles, respectively. Taken together, the comedone count (16,6 ± 4,2) in the outlier group was significantly lower (p<0,05) than the rest of the A subjects (24,4 ± 10,4).