Development of an Efficient Extraction Methodology to Analyse Potential Inflammatory Biomarkers from Sebum

Abstract

Introduction: Proteins, such as cytokines and chemokines, are present in varying concentrations in a range of biofluids, with an important signalling role in maintaining homeostasis. Commercial tapes have been employed to non-invasively collect these potential biomarkers in sebum from the skin surface to examine their concentrations in conditions including acne, atopic dermatitis and pressure ulcers. However, the identification of robust biomarker candidates is limited by the low abundance of specific proteins extracted by current methodologies. Therefore, this study was designed to develop an optimized extraction method for potential inflammatory biomarkers in sebum collected with Sebutapes. Methods: Commercial tapes (Sebutapes) coated with synthetic sebum were used to systematically evaluate the effects of chemical and mechanical stimuli on extraction efficiency. Varying concentrations of high and low abundance biomarkers (IL-1α, IL-6, Il-8, INF-γ, TNF-α and IL-1RA) were used to spike the synthetic sebum samples. Methodological variables included different surfactants, mechanical stimuli and buffer volume. Extraction efficiency was estimated using immunoassay kits from the extracted buffer. Results: The results revealed that the use of a surfactant, i.e. β-dodecyl maltoside in addition to the mechanical stimuli, namely sonication and centrifugation resulted in an increased recovery of cytokines, ranging from 80% for high-abundant cytokines, such as IL-1α and IL-1RA, and up to 50% for low-abundance cytokines, including TNF-alpha, IL-6 and IL-8. Compared to previous methods, the new extraction protocol resulted in between an 1.5 - 2.0 fold increase in extraction efficiency. Conclusion: The study revealed that there was a high degree of variability in the extraction efficiency of different cytokines. However, improved efficiency was achieved across all cytokines with selective surfactants and mechanical stimuli. The optimised protocol will provide means to detect low levels of potential biomarkers from skin surface, enabling the evaluation of local changes in pro- and anti-inflammatory cytokines present in different skin conditions.

The Effects of Incontinence Pad Application on Loaded Skin With Reference to Biophysical and Biochemical Parameters

“…Findings from a recent study indicated an increase in IL-1α when skin was in contact with pads moistened with 0.9% saline solution alone; however, no differences in the expression of low abundance proteins (TNF-α, IL-6, and IL-8) were detected 12. These discrepancies in study findings may also be attributed to our use of an optimized method of extraction involving both chemical and mechanical stimuli 29…”

“…12 These discrepancies in study findings may also be attributed to our use of an optimized method of extraction involving both chemical and mechanical stimuli. 29…”

Inflammatory biomarkers in sebum for identifying skin damage in patients with a Stage I pressure ulcer in the pelvic region: A single centre observational, longitudinal cohort study with elderly patients

“…Previous studies investigating the cytokines profiles following insults have reported changes in high‐abundant cytokines, such as IL‐1α and IL‐1RA 14,16 . However, with the use of the new extraction protocol, we were able to quantify low‐abundant cytokines, such as IL‐8 and G‐CSF that could provide a classification of damaged skin from healthy skin 22 . It is well known that a variety of skin insults (mechanical, chemical and thermal) alters the production of the high‐abundant cytokines, namely, IL‐1α and IL‐1RA 12 .…”

“… 14 , 16 However, with the use of the new extraction protocol, we were able to quantify low‐abundant cytokines, such as IL‐8 and G‐CSF that could provide a classification of damaged skin from healthy skin. 22 It is well known that a variety of skin insults (mechanical, chemical and thermal) alters the production of the high‐abundant cytokines, namely, IL‐1α and IL‐1RA. 12 In addition, each of the low‐abundant cytokines plays a unique role in maintaining the barrier function, for example, TNF‐α, INF‐γ and IL‐1β are important in lipid synthesis, whereas IL‐8 and G‐CSF are associated with dendritic cell migration and neutrophil regulation.…”

“…The extraction of skin inflammatory biomarkers was performed following an optimised protocol 22 with the use of chemical and mechanical stimuli to improve the extraction efficiency. To review briefly, the Sebutapes were extracted with 0.85 mL of extraction buffer, which consisted of PBS + 0.1% Dodecyl maltoside.…”