Chemicals and reagents
Acetonitrile (ACN), methanol, isopropanol (IPA), and Optima™ LC/MS Grade were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Sebutape was purchased from CuDerm Corporation (Dallas, TX, USA).
Participants
The study was reviewed and approved by the Ethics Committee of Peking University International Hospital. Informed consent to participate in the study was obtained from all patients (elderly with senile pruritus) and healthy individuals (elderly without pruritus) before enrollment. Individuals aged >60 years were eligible for inclusion. Forty participants from the Beijing area were enrolled in the study, including 20 patients with senile pruritus and 20 healthy controls. All 20 patients had physician-confirmed diagnosis of senile pruritus and were able to complete the study. The patients underwent a complete examination and checkup to exclude diabetes, liver or kidney dysfunction, malignant tumor, HIV infection, thyroid disorder, anxiety or depression disorder, psoriasis, atopic dermatitis, ichthyosis, scabies, eczema, bullous pemphigoid, and other dermatoses that can cause skin pruritus. The participants had not received any treatments or drugs that could interfere with the study assessment for 6 months, including cardiovascular, antiepileptic, antibiotic, and antipsychotic drugs. The participants did not bathe or apply topical moisturizer for 24 hours before the test. The study protocol ensured that the participants were matched for demographic characteristics of sex and age.
TEWL measurement
Transepidermal water loss (TEWL) measurements were performed at a site 1 cm below the right knee using a portable VapoMeter (TM300; CK, Cologne, Germany). The measurement time was 8–10 s. Briefly, the VapoMeter was maintained under standard ambient conditions in a cool air-conditioned room at temperature 23℃ and humidity 50%. After the detection probe was placed on the target area, three consecutive readings were collected from the same site and averaged for each participant.
SSL sampling
Before sample collection, participants were instructed to remain under standard ambient conditions (room temperature 23℃ and humidity 50%) in a cool air-conditioned room for 30 min. Sebum was collected from an approximately 4-cm2 area at the same site 1 cm below the right knee using Sebutape. Prior to sebum collection, the collection area was wiped with a 5% saline swab and one Sebutape patch was placed on the target site. The Sebutape patch was left in place for 10 min, and then removed to a sterile centrifuge tube using curved forceps. All samples were immediately stored at −80℃ until further analysis.
Itch intensity scale for assessment of pruritus severity
Pruritus severity in each patient was evaluated by the Ameliorated Kawashima Itch Scale (Supplementary Table 1) (10), which rated itch severity on a five-point scale (0, 1, 2, 3, 4) in separate diurnal and nocturnal assessments. The pruritus score was calculated by adding the diurnal score to the nocturnal score (range, 0–8).
Sample preparation
After retrieval of the samples from the −80°C freezer, 1.5 mL of chloroform/methanol (1:2 v/v) was added to each tube. The samples with chloroform and methanol were mixed by vortex vibration for 10min and placed at −20° for 12 h. Subsequently, 1.4 mL of supernatant was removed and 0.2 mL of chloroform was added to further extract the lipids. Each tube was then added with 1 mL of deionized water and centrifuged at 3000×g for 5 min to stratify the solution. After the stratification, the upper layer was the aqueous phase and the lower layer was the organic phase. A low temperature concentrator (Speed Vac SPD131P; Thermo Fisher Scientific) was used to dry the lipid extracts for later analysis. Before mass spectrometry detection, the lyophilized samples were dissolved in 100 µL of methanol/isopropanol (2:1 v/v). Sample mixtures were also prepared as quality control samples for analytical performance prior to analysis by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOFMS).
LC-MS/MS analysis and identification
Liquid chromatography was performed using LC instrument (Exion LC AD, AB SCIEX, USA: Exion LC AD, AB SCIEX, USA). LC separation was performed using a Phenomenex Kinetex 1.7u EVO C18 column (2.1×50 mm, 100A; Agilent, Santa Clara, CA, USA). The column temperature was 40℃ and the sample dosage was 3 μL. The flow rate was 0.5 mL/min. Mobile phase A was 50% water + 50% ACN + 10 mM ammonium formate and mobile phase B was 10% ACN + 90% IPA + 10 mM ammonium formate. The gradient elution conditions with mobile phase A and mobile phase B are shown in Table 1. MS was performed using a Triple TOF 5600+ (AB SCIEX, Concord, Ontario, Canada) and an orthogonal accelerated TOFMS equipped with an electrospray ion source. The ionization mode of positive and negative ions from an electrospray ionization source was used. The data collection method was TOF-MS-IDA-TOF-MS/MS (n=8). The quadrupole scanning range was m/z 50–1500. The detailed ion source conditions are shown in Table 2. An independent reference, Lock-mass ion, via the MS-Dial (ver. 3.70; 17 April 2019) was used to ensure mass accuracy during data acquisition.
The assigned modified metabolite ions were identified by database searches in the MS-Dial Lipidomics MSP database (http://prime.psc.riken.jb/compms/msdial/mail.html). The chromatographic retention behavior was considered to reduce false-positive matches (11).
Statistical analysis
Using MS-Dial (Ver. 3.70) and MS-Dial Lipidomics MSP database information transferred into the software, the original mass spectrum data were used for processes such as peak identification, peak filtering, and peak alignment. For multiple linear regression analysis, the results eventually generated a two-dimensional data matrix containing lipid name, peak time, lipid classification, mass core ratio (m/z), peak area, and original Excel file data. MetaboAnalyst 4.0 (http://www.metaboanalyst.ca/MetaboAnalyst/) was used to normalize the samples with different comparison requirements, and an Excel file called DATA_mart was obtained. A multivariate analysis, comprising partial least-squares discrimination analysis (PLS-DA), was constructed to determine the distributions and identify metabolic differences between the senile pruritus patients and healthy controls using MetaboAnalyst 4.0. The PLS-DA models were cross-validated using a 10-fold method with unit variance scaling. R2 evaluated the fitting of the PLS-DA model while Q2 evaluated the prediction ability. If the Q2 value was negative or extremely low, it meant that the differences between the two groups were not statistically significant. The PLS-DA model eliminated any changes in the X matrix that were not related to the Y matrix. Therefore, only one prediction component was used to distinguish the two classes.
Comparisons of the two groups related to the intensities of integrated regions were carried out using a two-tailed Welch’s t-test, which was performed within MetaboAnalyst 4.0. Values of p<0.05 were considered statistically significant.