Development of an improved method for quantitative analysis of skin blotting: increasing reliability and applicability for skin assessment

Abstract

OBJECTIVE:A novel skin assessment tool named "skin blotting" has been recently developed, which can easily predict the skin status to avoid its deterioration. The aim of this study is to propose a normalization method for skin blotting to compensate for individual differences that can hamper the quantitative comparisons and clinical applications. METHODS:To normalize individual differences, we utilized a total protein as a "normalizer" with calibration curves. For evaluation, we performed a simple simulation experiment, in which the same concentration of a protein of interest [tumor necrosis factor (TNF)-α] was applied at different volumes as a virtual individual difference. Moreover, to demonstrate the applicability of this normalization, male volunteers were recruited for skin blotting followed by the estimation of TNF-α with normalization. RESULTS:We obtained good calibration curves for total protein (R 2 = 0.995) and TNF-α (R 2 = 0.997), both of which were necessary for an exact quantification. In the simulation experiment,we estimated the exact concentration of TNF-α regardless of the applied volume, demonstrating the applicability of this normalization method in skin blotting. Further, skin blotting on human subjects showed a wide range of variation in the total protein content, although the normalization was thought to reduce such individual variations. CONCLUSION:This study has proposed total protein normalization for skin blotting with calibration curves. This method may strengthen the quantitative performance of skin blotting, which may expand the applicability of this method as a skin assessment tool in broader fields, such as nursing and cosmetology. reported that the skin of obese people is continuously exposed to oxidative stress, collagen-degrading enzymes [15], and inflammatory cytokines, e.g., tumor necrosis factor-α (TNF-α), all of which may be linked to skin weakness [16][17][18][19][20]. In another instance, skin maceration caused by incontinence leads to incontinence-associated dermatitis, which can be prevented by assessing the reduction of skin barrier function using a simple skin assessment tool [21]. From these findings, it may be important to assess the property of skin in a biochemical manner before the appearance of obvious drawbacks such as wound development that may affect the quality of life.Under these circumstances, several types of non-invasive skin assessment tools have been thus developed using laser analysis factor-β, and matrix metalloproteinase-2) in the dermis and epidermis can leak by diffusion through the epidermis and follicles when the skin is slightly macerated [18, 27]. In this method, a prewetted nitrocellulose membrane is patched to the region of interest on the skin, which creates a slightly macerated state. Under such a condition, the soluble proteins that leak from the epidermis and dermis can be captured by the membrane. The proteins on the membrane can then be analyzed using immunoblot techniques. This method is similar to the tape stripping method [2...

Increased level of tumour necrosis factor‐alpha (TNF‐α) on the skin of Japanese obese males: measured by quantitative skin blotting

“…Densitometric analyses with standard curves for total protein and TNF‐ α were performed using the ImageJ software (National Institute of Health, Bethesda, MD, USA). The linearity of standard curves for total protein and TNF‐ α was previously confirmed . To normalize the difference of protein adsorption in each subject, the amount of TNF‐ α was divided (i.e.…”

“…Quantitative skin blotting was performed to measure the level of TNF‐ α detectable on the skin, as described previously . In brief, a piece of nitrocellulose membrane (1 cm × 1 cm each; Bio‐Rad Laboratories, Hercules, CA, USA) was wetted with 50 μL of sterile normal saline (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan).…”

“…After 10 min, the membrane was removed and stored at 4°C until analyses. The membrane was first analysed using a total protein quantification . In brief, the skin blotting membranes, along with the membranes with known concentrations of the bovine serum albumin (Nacalai Tesque, Kyoto, Japan) for a calibration curve, were stained with 3% Reactive Brown 10 (Sigma–Aldrich, MO, USA).…”

“…With a normalization method, quantitative skin blotting can be utilized for skin protein quantification. As a preliminary result of normalization experiment, the levels of TNF‐ α detected on the skin were significantly higher in the obese (BMI ≥ 30) subjects than in the normal (BMI < 25) subjects , which led us to conduct the detailed analyses between obesity and TNF‐ α on the skin.…”

“…In that study, they found no correlation between the body mass index (BMI) and the immunoreactivity of blotted TNF‐ α ; this might be because a normalization method, which eliminates individual differences due to the protein adsorption, attachment force, skin conditions, etc., was not used. For quantification of skin blotting, a normalization method was developed . With a normalization method, quantitative skin blotting can be utilized for skin protein quantification.…”