Jayabal Hemalatha, Bader Dan L, Worsley Peter
School of Health Sciences, University of Southampton, Southampton, UK.
Skin Pharmacol Physiol. 2023;36(1):38-50. doi: 10.1159/000528653. Epub 2022 Dec 26.
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.
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.
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-α, IL-6 and IL-8. Compared to previous methods, the new extraction protocol resulted in between a 1.5-2.0-fold increase in extraction efficiency.
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.
蛋白质，如细胞因子和趋化因子，以不同浓度存在于一系列生物流体中，在维持体内平衡方面发挥着重要的信号传导作用。商业胶带已被用于从皮肤表面无创收集皮脂中的这些潜在生物标志物，以检测它们在痤疮、特应性皮炎和压疮等病症中的浓度。然而，由于当前方法提取的特定蛋白质丰度较低，难以鉴定出可靠的生物标志物候选物。因此，本研究旨在开发一种优化的提取方法，用于从使用皮脂胶带收集的皮脂中提取潜在的炎症生物标志物。
使用涂有合成皮脂的商业胶带（皮脂胶带）系统评估化学和机械刺激对提取效率的影响。将不同浓度的高丰度和低丰度生物标志物（IL-1α、IL-6、IL-8、INF-γ、TNF-α和IL-1RA）添加到合成皮脂样品中。方法变量包括不同的表面活性剂、机械刺激和缓冲液体积。使用从提取缓冲液中提取的免疫分析试剂盒评估提取效率。
结果显示，除了机械刺激（即超声处理和离心）外，使用表面活性剂β-十二烷基麦芽糖苷可提高细胞因子的回收率，高丰度细胞因子（如IL-1α和IL-1RA）的回收率为80%，低丰度细胞因子（包括TNF-α、IL-6和IL-8）的回收率高达50%。与以前的方法相比，新的提取方案使提取效率提高了1.5至2.0倍。
该研究表明，不同细胞因子的提取效率存在高度变异性。然而，通过选择性表面活性剂和机械刺激，所有细胞因子的提取效率均得到提高。优化后的方案将提供检测皮肤表面低水平潜在生物标志物的方法，从而能够评估不同皮肤病症中促炎和抗炎细胞因子的局部变化。
