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Quantitative Analysis of Complex Lipids in Human Sebum
Tracy Shafizadeh, PhD and Steve Watkins, PhD | Lipomics, A Division of Metabolon
introduction
Sebum is a complex mixture of lipids secreted by mature sebocytes onto the surface of
skin. Sebum may have antimicrobial, photoprotection and vitamin delivery functions, and
changes in the concentration and composition of sebum are related to acne and other
skin disorders. Sebum is comprised of an unusual mix of lipid classes, distinct from other
biological matrices such as plasma, liver or adipose. Further, the fatty acids comprising
the complex lipid classes are largely unique to skin metabolism and include odd and
branched chain fatty acids, and a high concentration of the unusual sapienic acid
(16:1n10) and its metabolites.
Here we describe a platform for fully quantitative, high throughput compositional
analysis of complex lipids in human sebum extracted from Sebutape®. The TrueMass™
Sebum Lipid Panel measures the concentration and fatty acid composition (40 fatty
acids) of triacylglycerol, wax ester, free fatty acid and diacylglycerol as well as squalene
from 3 pooled Sebutapes. The panel is useful for monitoring the effect of drug, cosmetic
and nutritional interventions on the quality and quantity of sebum in human subjects and
cell or animal models.
Figure 2 shows the concentration of the major lipid classes found in human sebum, expressed
per Sebutape, in healthy control subjects (n=15). TG data are expressed as nMol fatty acid,
rather than total amount of lipid class, to highlight the abundance of the TG class compared to
all other classes.
METHODS
Sebum was collected for thirty minutes from the forehead region of healthy, untreated
subjects using Sebutape skin patches (CuDerm Corp, Dallas, TX). For each sample, three
patches are pooled and extracted by sonication in ethanol for five minutes. The extract
is dried under nitrogen and re-dissolved in hexanes. An aliquot of the extract is taken for
squalene analysis and the remainder is passed through a silicon solid phase cartridge.
Sebum lipids are eluted from the solid phase cartridge with 2mL of hexanes followed
by 5mL ethyl ether: chloroform: hexanes. Samples are dried under nitrogen, and then
separated via thin layer chromatography. Isolated lipid classes are trans-methylated
in HCl: methanol and then isolated in hexane. Samples are injected onto GC-FID for
analysis. The absolute concentration of each fatty acid in each lipid class is determined
by comparing the peak to that of the relevant internal standard.
The squalene aliquot is dried under nitrogen and dissolved in 1M KOH in ethanol.
Samples are saponified for 1 hour at 70 degrees C. Squalene is isolated in hexanes,
dried, and derivatized in Tri-sil. Samples are injected onto GC-MS for analysis. The
absolute concentration of squalene acid is determined by comparing its peak to that
of an internal standard.
Figure 3 shows the mol% concentration of individual fatty acids within each lipid class found in
sebum from healthy control subjects (n=15). The distribution of fatty acids varied significantly
among all lipid classes.
Panel Markers
TrueMass Sebum Lipid panel measures the absolute concentration (nMoles/gram) of:
triacylglycerol (TG), wax ester (WE), diacylglycerol (DG), free fatty acids (FA) and squalene.
The relative abundance of fatty acids in each lipid class (other than squalene), as
mole% composition, is reported and includes the following fatty acids: 12:0, 13:0, 14:0,
15:0, 16:0, 17:0, 18:0, 20:0, 21:0, 22:0, 23:0, 24:0, 25:0, 26:0, 27:0, 28:0, 29:0, 30:0, 12:0-iso,
13:0-iso, 14:0-iso, 15:0-iso, 16:0-iso, 18:0-iso, 20:0-iso, 21:0-iso, 13:0-anteiso, 15:0-anteiso,
17:0-anteiso, 19:0-anteiso, 21:0-anteiso, 16:1n7, 16:1n10, 18:1n7, 18:1n9, 18:1n10, 18:1n12,
18:2n6, 18:2n10 and 18:3n3.
results
Figure 4 shows Pearson correlation coefficients among all lipid analytes displayed in a correlation
matrix. The results suggest there is complexity to the metabolic regulation of sebum lipids that can
be interrogated using this platform.
CONCLUSION
We describe here a fully quantitative, high throughput method for lipid profiling of
human sebum from Sebutape. This method allows for detailed assessment of sebum
lipid composition. Understanding the unique composition and metabolic regulation of
sebum lipids provides insight into improved biomarker identification and therapeutic
development for a variety of skin conditions.
Figure 1 provides a representative chromatogram of quantitative lipid profiling of human sebum,
from three pooled Sebutapes.