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MB Sample ID: SA314363
Local Sample ID: | LN949 |
Subject ID: | SU002987 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002987 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
LN949 | SA314363 | FL037439 | LN | Sample_group |
LN949 | SA314363 | FL037439 | All class V | Disease_subclass |
Collection:
Collection ID: | CO002980 |
Collection Summary: | Both morning urine and blood samples were collected under sterile conditions within the same day of biopsy/recruitment. Urine samples were centrifuged at 3,500 rpm for 10 min at 4°C. The supernatant was then aliquoted and stored at −80°C until the analysis. Common blood biochemical parameters were measured by an ISO 15189 accredited laboratory. Serum creatinine (SCr) and urine creatinine (UCr) were measured by an enzymatic assay using a Dimension ExL analyzer (Siemens Healthcare Diagnostics, Newark, DE, USA). Urine protein (Uprot) was measured by a modified pyrogallol red-molybdate method. Uprot was reported as urine protein creatinine ratio (UPCR in mg/mgCr). The estimated glomerular filtration rate (eGFR in mL/min/1.73 m2) was calculated using the CKD-EPI equation (Levey et al., 2009): |
Sample Type: | Urine |
Treatment:
Treatment ID: | TR002996 |
Treatment Summary: | LN patients and health subjects (N) were recruited from Ramathibodi Hospital, Bangkok, Thailand. Patients, who fulfilled at least four of the American College of Rheumatology 1982 revised criteria for SLE (Hochberg, 1997) and were referred to kidney biopsy for clinical indications of proteinuria ≥ 0.5g/24 hours (Fanouriakis et al., 2020) were included. |
Sample Preparation:
Sampleprep ID: | SP002993 |
Sampleprep Summary: | The urine samples were prepared based on a previously published protocol (Zhu et al., 2019) with minor modifications. Briefly, a 50 μL of each urine sample was mixed with 200 μL of MeOH/ACN (1:1, v/v) containing 100 ng of anthranilic acid C13 (Ant-C13) as an internal standard (IS). The mixture was vortexed for 30 s and sonicated for 10 min (room temperature). The mixture was then left overnight (−20°C) for protein precipitation before centrifugation at 13,000 rpm (4°C) for 15 min. The supernatant was transferred to a new test tube and evaporated to dryness (room temperature) using a vacuum concentrator (Labconco, MO, USA). The dried sample was reconstituted in 100 μL of Milli-Q H2O (containing 0.1% formic acid), vortexed for 30 s, and sonicated (room temperature) for 10 min. The reconstituted sample was centrifuged at 13,000 rpm (4°C) for 15 min. The supernatant was kept at −80°C before analysis. |
Combined analysis:
Analysis ID | AN004711 |
---|---|
Analysis type | MS |
Chromatography type | Normal phase |
Chromatography system | Waters Acquity I-Class |
Column | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | Waters Xevo TQ-S |
Ion Mode | POSITIVE |
Units | ng/ml |
Chromatography:
Chromatography ID: | CH003547 |
Chromatography Summary: | A volume of 5 μL of a standard or sample was injected onto an HSS T3 column, 2.1 × 100 mm, 1.8 μM column (Waters, Milford, MA, USA) at 30°C with a constant flow rate of 0.3 mL/min. Mobile phases consisted of (A) 0.1% formic acid in Milli-Q water and (B) 0.1% formic acid in ACN. The gradient program started at 99% A, decreased to 70% over 7.0 min, then decreased to 30% at 9 min, and return to the initial condition at 10 min and held for 4.0 min. The total runtime was 14 min. The weak and strong needles wash solutions were 5% ACN and H2O/ACN/MeOH/IPA (1:1:1:1), respectively. |
Instrument Name: | Waters Acquity I-Class |
Column Name: | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
Column Temperature: | 30 |
Flow Gradient: | The gradient program started at 99% A, decreased to 70% over 7.0 min, then decreased to 30% at 9 min and return to the initial condition at 10 min and held for 4.0 min. The total runtime was 14 min. The weak and strong needles wash solutions were 5% ACN and H2O/ACN/MeOH/IPA (1:1:1:1), respectively. |
Flow Rate: | 0.4ml/min |
Solvent A: | 100% water; 0.1% formic acid |
Solvent B: | 100% acetonitrile; 0.1% formic acid |
Chromatography Type: | Normal phase |
MS:
MS ID: | MS004457 |
Analysis ID: | AN004711 |
Instrument Name: | Waters Xevo TQ-S |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | Quantitative analysis (absolute quantification) was achieved by external calibration curves of each standard prepared in pooled urine samples (standard addition) (Limjiasahapong et al., 2021). The linear ranges of the calibration standards are as follows: 3.5–1,000 ng/mL for Ant; 4.5–80 ng/mL for Cin; 8–2,500 ng/ml for Kyna; 8–5,000 ng/ml for Kyn; 1.4–400 ng/ml for Pic; 40–5000 ng/ml for Qui; 7–2000 ng/ml for Trp; 2–200 ng/ml for Xan; 900–14,000 ng/ml for 3OH-Kyn; 300–2,500 ng/ml for 3OH-Ant and 8–2,000 ng/ml for Ant-C13. |
Ion Mode: | POSITIVE |