| Objective:Acne vulgaris is an inflammatory skin disease involving pilosebaceous units, with a high incidence, could be mainly seen in adolescence. Severe acne could lead to permanent scarring, bringing patients a double blow economically and psychologically. There is still no safe and effective treatment for acne, thus further researches on the mechanism of acne, which would find more economical, effective and safe treatment is an urgent problem to be solved.Acne vulgaris is a multifactorial and polygenic disease, currently there are four accepted pathogenesis: androgen-induced increasing sebum secretion, abnormal keratinization, inflammation, as well as bacteria(P. acnes: Propionibacterium acnes) colonization in the hair follicles involving face, neck, chest and back. Recent researches have also confirmed that the composition of sebum is the major cause of acne. Linoleic acid is the most common essential fatty acid in skin, a precursor of ceramide, plays an important role in maintaining epidermal homeostasis and permeability barrier of the stratum corneum. Squalene content is higher in acne patients, squalene oxide could promote epidermal cells release inflammatory cytokines, thus speeding up the acne formation.TLC is the most common method in previous studies, the researches on HPLC determination of sebum composition is rare. We found a HPLC method for simultaneous determination of the content of linoleic acid and squalene in human sebum, in order to establish a convenient and highly sensitive method. By comparing the total sebum, linoleic acid and squalene content between acne patients and normal people, to further explore the significance of sebum and free fatty acids in the pathogenesis of acne, thus provide the theoretical basis for the safer and more effective treatment. Methods:Research objects including 60 cases of acne vulgaris patients from the Dermatology outpatient in the first Affiliated Hospital of Fujian Medical University, consistent with the diagnosis standards of "Chinese Journal of Clinical Dermatology(first edition)" and Pillsbury taxonomy of grade II, III, IV. All cases of acne vulgaris patients without anti-acne treatment in the last three months, including oral and topical medications. Another 62 cases of normal volunteers, were the Students from Fujian Medical University, all subjects excluded from other factors and systemic diseases that could affect sebum secretion, and allergic to alcohol.Firstly, establishing a HPLC method for simultaneous determining content of linoleic acid and squalene in human sebum, including standard solution preparation, chromatographic conditions exploration, specificity experiments, standard curve preparation, recoveries and precision experiments. The specimens were collected, weighed, handled, after linoleic acid and squalene were extracted by n-hexane solution and purified by gradient elution with reverse phase column, then analyzed the content of linoleic acid and squalene in both groups at 210 nm. For the statistical analysis of measurement data, if the data followed a normal distribution, the difference between the groups used t test, if the data is not normal, the Wilcoxon test was used to compare the differences between groups. For statistical analysis of count data, the difference between the groups used ?2 test.Results: 1. In this study, linoleic acid and squalene could completely be separated, the retention time were 7.007 min and 22.847 min, impurity of mobile phase and the filter papers did not affect linoleic acid and squalene determination. Linoleic acid had a good linear relationship in the range of(53.4~1708.8)?g·m L-1; Squalene had a good linear relationship in the range of(46.8~5616.0)?g·m L-1; Recoveries were 90%~110%; Precision(intra and inter-day) were(-15%~+ 15%). 2. The comparison of sebum weight: sebum weight both in normal group and acne group were higher in men(p<0.05); sebum weight both in women and men were higher in acne group(p<0.05). 3. The comparison of linoleic acid and squalene content: linoleic acid levels both in women and men were lower in acne group(p<0.05); squalene levels both in women and men were higher in acne group(p<0.05).Conclusion: The established HPLC method for simultaneous determining content of linoleic acid and squalene has the characteristics of high sensitivity, easy manipulation, rapidity and accuracy. The pathogenesis of acne vulgaris is related to elevated levels of sebum secretion, decreased content of linoleic acid, and increased content of squalene. |
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