Chemicals and reagents
Methanol, chloroform, ammonium formate, isopropyl alcohol, acetonitrile, formic acid, and distilled water used during sample preparation and LC-MS/MS were purchased from Thermo Fisher Scientific (Waltham, MA, USA). SSL-adsorbent tape (Sebutape) was purchased from CuDerm Corporation (Dallas, TX, USA).
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. A total of 40 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 receive 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.
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.
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).
Samples were retrieved from the −80°C freezer, and 1.5 mL of reagent mixture (chloroform/methanol) was added. The samples were vortexed and allowed to settle for 30 min. An equal volume of chloroform was then added, and the samples were vortexed and allowed to settle for 10 min. The lipid extracts were dried using a low-temperature concentrator (SpeedVac SPD131P; Thermo Fisher Scientific). Finally, the lipids were redissolved in a reagent mixture (methanol/isopropyl alcohol). Before analysis by ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry(UPLC-QTOFMS), a mixture of samples was prepared for use as quality control samples for the analytical performance.
LC-MS/MS analysis and identification
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 was maintained at 40℃. The injected sample volume was 3 μL for each run in the full loop injection mode. The flow rate of the mobile phase was 0.5 mL/min. In the reverse-phase LC mode, gradient elution was performed with the following solvent system: (A) 50% acetonitrile (Optima™ LS/MS grade; Fisher) in water and 10 mM ammonium formate; (B) isopropyl alcohol with 10% formic acid and 10 mM ammonium formate. The gradient started with 90% A, decreased to 0% A in 11 min, was held at 100% B for 6 min, immediately returned to 90% A, and was held at 90% A for 3 min. MS was performed using a Triple TOF 5600+ (AB SCIEX, Concord, Ontario, Canada), an orthogonal accelerated time-of-flight mass spectrometer equipped with an electrospray ion source. Data were acquired in the positive and negative V-geometry mode for each chromatography separation in the LC-MS/MS analysis. The capillary voltages were set to 5500 V and −4500 V, cone gas at 50 L/h, desolvation gas at 600 L/h, and source temperature at 550℃. The scan range was m/z 50 to m/z 1200 in the full scan mode and data were collected in the IDA mode. 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 databases(http://prime.psc.riken.jb/compms/msdial/mail.html). The chromatographic retention behavior was considered to reduce false-positive matches (11).
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 (http://www.metaboanalyst.ca/MetaboAnalyst/). The PLS-DA models were cross-validated using a 10-fold method with unit variance scaling. R2 was used to evaluate the fitting condition of the PLS-DA models, and Q2 was used to assess the predictive ability. Negative or very low Q2 values indicated that the differences between the groups were not statistically significant. The PLS-DA models removed any variation in the X matrix that was not correlated with the Y matrix. Thus, only one predictive component was normally used for discrimination between 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.