Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA226606

Local Sample ID:	B33
Subject ID:	SU002388
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	>=18
Gender:	Male and female
Human Race:	Chinese
Human Ethnicity:	Han
Human Trial Type:	observational study
Human Medications:	Dupilumab
Human Inclusion Criteria:	1.Age ≥ 18 years of age 2.Dermatologist diagnosis of moderate to severe AD, EASI≥16 at baseline 3.Eligible to receive dupilumab therapy for AD in accordance with the guidelines. Patients who are eligible were treated with a fixed schedule of 300mg dupilumab in 2-week intervals. Patients who did not achieve 16-week therapy were excluded. 4.During the whole treatment process, the requirements for diet and exercise are roughly the same as before treatment, so as to keep the body healthy and balanced 5.A 30-day washout period of systemic medications preceded treatment
Human Exclusion Criteria:	1Evidence of other skin diseases except for AD at baseline 2.Pregnancy or breast feeding, 3.Patients with permanent severe diseases, especially those affecting the immune system, except asthma 4.Patients with severe mental illness 5.Evidence of chronic metabolic disease, including Obesity, diabetes, fatty liver, osteoporosis, atherosclerotic cardiovascular and cerebrovascular diseases, and metabolic-related cancers (breast, colorectal, pancreatic, colon, and prostate cancer). 6.Application of other systemic medications during treatment

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Sample Preparation:

Sampleprep ID:	SP002394
Sampleprep Summary:	Metabolomics_LC-MS 100 μL of sample was transferred to an EP tube. After the addition of 400 μL of extract solution (acetonitrile: methanol = 1: 1, containing isotopically-labelled internal standard mixture), the samples were vortexed for 30 s, sonicated for 10 min in ice-water bath, and incubated for 1 h at -40 ℃ to precipitate proteins. Then the sample was centrifuged at 12000 rpm(RCF=13800(×g),R= 8.6cm) for 15 min at 4 ℃. The resulting supernatant was transferred to a fresh glass vial for analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples. Metabolomics_GC-MS Transfer 50 μL sample to EP tube and add 205 μL precooled extract methanol，(including internal L-2-Chlorophenylalanine, 1mg/mL stock), vortex mixing for 30 s. Ultrasound for 10 min (ice bath) . After centrifugation at 4 ℃ for 15 min at 12000 rpm(RCF=13800(×g),R= 8.6cm) . Carefully transfer the 180μL supernatant into a 1.5 mL EP tube. Take 50 μL of each sample and mix them into QC samples. Dry extract in vacuum concentrator. After evaporation in a vacuum concentrator, 30 μL of Methoxyamination hydrochloride (20 mg/mL in pyridine) was added and then incubated at 80 ℃ for 30 min, then derivatized by 40 μL of BSTFA regent (1% TMCS, v/v) at 70 ℃ for 1.5h. Gradually cooling samples to room temperature, 5 μL of FAMEs (in chloroform) was added to QC sample. All samples were then analyzed by gas chromatograph coupled with a time-of-flight mass spectrometer (GC-TOF-MS). Lipidomics 100 μL of sample was transferred to an EP tube, and added with 480 μL of extract solution (MTBE: methanol1 = 5: 1). After 30 s vortex, the samples were sonicated for 10min in ice-water bath, incubated at -40 ℃ for 1 h, and centrifuged at 3000 rpm (RCF=900(×g),R= 8.6cm) for 15 min at 4 ℃. r l.a$quchu1` μL of supernatant was transferred to a fresh tube and dried in a vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted in 100 μL of 50% methanol in dichloromethane. After 30s vortex, the samples were sonicated for 10 min in ice-water bath. The constitution was then centrifuged at 13000 rpm (RCF=16200(×g),R= 8.6cm) for 15 min at 4 ℃, and 75 μL of supernatant was transferred to a fresh glass vial for LC/MS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot 20 μL of the supernatants from all of the samples.

Sampleprep ID:

SP002394

Sampleprep Summary:

Metabolomics_LC-MS 100 μL of sample was transferred to an EP tube. After the addition of 400 μL of extract solution (acetonitrile: methanol = 1: 1, containing isotopically-labelled internal standard mixture), the samples were vortexed for 30 s, sonicated for 10 min in ice-water bath, and incubated for 1 h at -40 ℃ to precipitate proteins. Then the sample was centrifuged at 12000 rpm(RCF=13800(×g),R= 8.6cm) for 15 min at 4 ℃. The resulting supernatant was transferred to a fresh glass vial for analysis. The quality control (QC) sample was prepared by mixing an equal aliquot of the supernatants from all of the samples. Metabolomics_GC-MS Transfer 50 μL sample to EP tube and add 205 μL precooled extract methanol，(including internal L-2-Chlorophenylalanine, 1mg/mL stock), vortex mixing for 30 s. Ultrasound for 10 min (ice bath) . After centrifugation at 4 ℃ for 15 min at 12000 rpm(RCF=13800(×g),R= 8.6cm) . Carefully transfer the 180μL supernatant into a 1.5 mL EP tube. Take 50 μL of each sample and mix them into QC samples. Dry extract in vacuum concentrator. After evaporation in a vacuum concentrator, 30 μL of Methoxyamination hydrochloride (20 mg/mL in pyridine) was added and then incubated at 80 ℃ for 30 min, then derivatized by 40 μL of BSTFA regent (1% TMCS, v/v) at 70 ℃ for 1.5h. Gradually cooling samples to room temperature, 5 μL of FAMEs (in chloroform) was added to QC sample. All samples were then analyzed by gas chromatograph coupled with a time-of-flight mass spectrometer (GC-TOF-MS). Lipidomics 100 μL of sample was transferred to an EP tube, and added with 480 μL of extract solution (MTBE: methanol1 = 5: 1). After 30 s vortex, the samples were sonicated for 10min in ice-water bath, incubated at -40 ℃ for 1 h, and centrifuged at 3000 rpm (RCF=900(×g),R= 8.6cm) for 15 min at 4 ℃. r l.a$quchu1` μL of supernatant was transferred to a fresh tube and dried in a vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted in 100 μL of 50% methanol in dichloromethane. After 30s vortex, the samples were sonicated for 10 min in ice-water bath. The constitution was then centrifuged at 13000 rpm (RCF=16200(×g),R= 8.6cm) for 15 min at 4 ℃, and 75 μL of supernatant was transferred to a fresh glass vial for LC/MS analysis. The quality control (QC) sample was prepared by mixing an equal aliquot 20 μL of the supernatants from all of the samples.