Detection steps
The detailed detection steps are as follows: First, Attach the Teflon gasket with a detection well in the middle to the small thenar part of the palm; Then, Add 100 μl of detection reagent to the detection well, after a 1-min incubation, the reagents are removed by blotting and the 30 μl of TMB is added to the well, after an additional 2-min incubation, 30 μl of the reaction solution is transferred to the sample pool with pipettor on the sample platform. Finally, the value of cholesterol content is acquired according to the absorption spectrum of the reaction solution.
Skin cholesterol detection reagent
The detection reagent is digitonin–copolymer–horseradish peroxidase (HRP) conjugate, which has beed proposed as the specific reagent for skin cholesterol detection[9], in simple terms, digitonin have strong affinity with cholesterol, and it can combine with cholesterol to form cholesterol-digtonin complex[19-21], the copolymer ensures a tight bond between digtonin and horseradish peroxidase, and the horseradish peroxidase can catalyze the color change of 3, 3’, 5, 5.-tetramethylbenzidine (TMB) substrate, which is dependent on the amount of cholesterol bound by the detection reagent. The reagent was synthesized following the steps described in the patent (authorization number: US05489510), TMB chromogenic reagent was purchased from Sigma-Aldrich (Shanghai, China). The detection reagent was diluted to 5 μg/ml , 2.5 μg/ml, 1.25 μg/ml, 0.625 μg/ml, 0.3125 μg/ml and 0.15625 μg/ml with deionized water to catalyze with TMB, the amount of colored products produced by the reaction were measured with our system.
Gradient Cholesterol extraction from porcine skin
Abdominal skin samples were obtained from six Tibetan pigs weighing 30-35 kg, and were provided by the animal center of Anhui medical university. Normal saline (shanghai, sigma) was used to soak the skin after subcutaneous tissue was removed so as to exclude hemoglobin and other pollution. The skin was then dried in the shade of the nature after cutting into 1.5*7.5 cm rectangular cubes. Skin cholesterol was extracted afterward in the mixture of ethanol and ethyl ether with a proportion of 3:1 for different time course (0 min, 1 min, 2 min, 3 min and 4 min) to obtain skin containing gradient cholesterol.
Participants
26 patients (45-80 years of age) diagnosed with atherosclerosis according to clinical information and coronary arteriography or carotid artery ultrasonography analysis were enrolled in The First Affiliated Hospital of University of Science and Technology of China from February to May 2019. Extent of disease was defined as the number of vessels with ≥50% stenosis and patients were further classified as having angiographic disease if the extent of disease was at least “one”. 79 high risk population(>10) and 58 low risk individuals(≤10) assessed by Framingham scoring from Health Management Center of Renmin Hospital of WuHan University were enrolled as control groups. Meanwhile, another 72 volunteers were recruited to participate in the accuracy verification, briefly, everyone involved in this experiment would measure skin cholesterol with non-invasive detection system, then the detection site will extracted with 400 ul of absolute ethanol for 2 minutes, cholesterol in extractive liquid were determined with gas chromatography immediately. Exclusion criteria included current use of cholesterol lowering medications, hepatitis, pregnancy, or skin disease on either hand.
Cholesterol measurement with gas chromatography
Cholesterol standard (Sigma-Aldrich) is dissolved in absolute ethanol to the concentration of 1 ug/ml, 2 ug/ml, 5 ug/ml, 10 ug/ml, 25 ug/ml and 50 ug/ml. Gas chromatography is used for detection of cholesterol content followed the previously reported method[22], briefly, detection condition is as follows: Column, DB-5 elastic quartz capillary column. Carrier gas, high purity nitrogen, purity ≥ 99.999 %; Constant flow rate, 2.4 mL/min; Column temperature (programming temperature): initial temperature is 200 ° C, hold for 1 min, increase to 280 ° C at 30 ° C / min, for 10 min; Inlet temperature, 280 °C; Detector temperature, 290 °C; Injection volume: 1ul; Injection method: no split injection, open the valve after 1 minute of injection; Air flow: 350 mL / min; Hydrogen flow rate: 30 mL/min. The cholesterol standard solution was separately injected into the gas chromatograph, and the peak area of the standard solution was measured under the above chromatographic conditions, and the standard curve was prepared by taking the concentration as the abscissa and the peak area as the ordinate. Then the extract was injected into the gas chromatograph to measure the peak area, and the concentration of cholesterol in the sample solution was obtained from a standard curve.
Statistic analysis
One-way ANOVAs (Graphpad, Prism 5) were utilized for multiple-group comparisons. All analysis was presented as means ± SD, A P value < 0.05 was considered statistically significant.