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MB Sample ID: SA078306
| Local Sample ID: | Prednisolone-d0-P3 |
| Subject ID: | SU001204 |
| Subject Type: | Mammal |
| Subject Species: | Canis lupus familiaris |
| Taxonomy ID: | 9615 |
| Genotype Strain: | Beagle |
| Age Or Age Range: | 8 to 83 months |
| Weight Or Weight Range: | 12.2 to 18.9 kg |
| Gender: | Male and female |
| Animal Animal Supplier: | In-house breeding |
| Animal Feed: | Standard pellet/kibble maintenance diet |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001204 |
| Subject Type: | Mammal |
| Subject Species: | Canis lupus familiaris |
| Taxonomy ID: | 9615 |
| Genotype Strain: | Beagle |
| Age Or Age Range: | 8 to 83 months |
| Weight Or Weight Range: | 12.2 to 18.9 kg |
| Gender: | Male and female |
| Animal Animal Supplier: | In-house breeding |
| Animal Feed: | Standard pellet/kibble maintenance diet |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Prednisolone-d0-P3 | SA078306 | FL011943 | Prednisolone | TreatmentGroup |
| Prednisolone-d0-P3 | SA078306 | FL011943 | 0d | TreatmentDuration |
| Prednisolone-d0-P3 | SA078306 | FL011943 | before | SamplingTimePoint |
Collection:
| Collection ID: | CO001198 |
| Collection Summary: | Blood was collected from the jugular vein after overnight fasting right before start and at the end of the treatments (12 h after the last prednisolone administration and 25 weeks after start of tetracosactide infusion, respectively). Plasma was prepared lithium heparin anticoagulated blood (Vacuette Greiner Bio-One, Germany) that centrifuged immediately after collection (1,862 g, 10 min, 4°C). Plasma samples were stored at −80°C until lipidomic analysis. |
| Collection Protocol Comments: | Blood collected after overnight fasting |
| Sample Type: | Blood (plasma) |
| Collection Method: | Venepuncture |
| Collection Location: | Jugular vein |
| Collection Frequency: | Before and after treatment |
| Storage Conditions: | -80℃ |
| Collection Vials: | VACUETTE® lithium heparin tubes (Greiner Bio-One, Germany) |
| Collection Tube Temp: | Room temperature |
| Additives: | Lithium heparin |
Treatment:
| Treatment ID: | TR001219 |
| Treatment Summary: | In the short-term prednisolone group, dogs were treated with 50 mg prednisolone (Streuli Pharma AG, Switzerland) orally twice daily for 3 consecutive days. For the long-term tetracosactide treatment, ALZET osmotic pumps (Durect Corporation, USA) subcutaneously delivering tetracosactide (synthetic ACTH; Bachem AG, Switzerland) were implanted into the dorsolateral neck. New pumps were implanted every 4 weeks. Total infusion time was 25 weeks, with a starting dose of 1.3–1.95 µg/kg/d tetracosactide, which was gradually increased to a final dose of 6–10 µg/kg/d. |
Sample Preparation:
| Sampleprep ID: | SP001212 |
| Sampleprep Summary: | Plasma lipids were extracted using a single-phase butanol/methanol extraction method (Alshehry et al, Metabolites 2015 with modifications). After thawing on ice, a 10 µL aliquot of each plasma sample was transferred into a 2 mL polypropylene tube (Eppendorf, Germany). Subsequently, 1 µL BHT (2,6-di-tert-butyl-4-methylphenol, 10 mmol/L in ethanol) and 90 µL of 1-butanol:methanol (1:1, v/v) containing internal standards was added to each sample. Following internal standards were added at 50 ng/mL final concentration in the extraction solvent: ceramide d18:1/17:0 (Avanti 860517P), Glucosylceramide (Matreya 1533), Lactosylceramide d18:1/16:0 D3 (Matreya 1534), LPC 20:0 (Avanti 855777P), LPE 14:0 (Avanti 856735P), PI 12:0/13:0 (Avanti LM-1500), PE 14:0/14:0 (Avanti 850745P), PS 14:0/14:0 (Avanti 840033P), SM d18:1/12:0 (Avanti 860583P), and at 100 ng/mL: DG 12:0_12:0 (Avanti 800812P), PC 14:0/14:0 (Avanti 850345P), TAG 16:0_16:0_16:0 d5 (CDN Isotopes D5815). Samples were then vortexed (30 sec) and sonicated in an ultrasound water bath for 30 min (20°C). After centrifugation (14,000 g, 10 min, 4°C), 90 µL of supernatant were transferred into 1.5 mL polypropylene tubes (Sarstedt, Germany) and dried under a nitrogen stream at 37°C. Dried extracts were stored at −80°C until LC-MS analysis. Dried samples were reconstituted with 90 µL 1-butanol:methanol (1:1, v/v) and sonication in an ultrasound water for 10 min. After centrifugation for 10 min at 20,800 g (4°C), supernatants (80 µL) were transferred into autosampler vials with glass inserts (Agilent Technologies, USA). For the analysis of sphingosine-1-phosphate (S1P), lipid extracts were derivatized according to the method described by Narayanaswamy et al. (Anal. Chem., 2014). To 50 µL lipid extract (see above), 50 µL 13C2D2–S1P d18:1 internal standard solution (Toronto Research Chemicals, Canada; 20 ng/mL in 1-butanol:methanol 1:1 [v/v]) was added. Derivatization was performed by adding 20 µL TMS-diazomethane (2 mol/L in hexanes; Acros Organics, Thermo Fisher Scientific, USA) and subsequent incubation for 20 min at 25°C and 700 rpm (Thermomixer, Eppendorf, Germany). To stop the reaction and inactivate TMS, 1 µL acetic acid 100% (glacial) was added. After centrifugation for 10 min at 20,800 g (7°C), supernatants were transferred into autosampler vials for LC-MS analysis. Process Quality Control (PQC) samples were generated by pooling equal volumes of plasma samples within an experimental group. For Blank samples, plasma was omitted. PQC and Blank were processed together with the study plasma samples with the same procedures. |
| Extraction Method: | Liquid–liquid extraction using butanol:methanol |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN001870 | AN001871 | AN001872 | AN001873 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | HILIC | Reversed phase |
| Chromatography system | Agilent 1290 Infinity | Agilent 1290 Infinity | Agilent 1290 Infinity | Agilent 1100 |
| Column | Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å) | Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å) | Waters Acquity BEH HILIC (100 x 2.1mm,1.7um,130 Å) | Agilent Zorbax Eclipse XDB C18 (150 x 3.0mm,1.8um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | Triple quadrupole | Triple quadrupole | Triple quadrupole | Triple quadrupole |
| MS instrument name | Agilent 6460 QQQ | Agilent 6495 QQQ | Agilent 6490 QQQ | ABI Sciex 4000 QTrap |
| Ion Mode | POSITIVE | POSITIVE | POSITIVE | POSITIVE |
| Units | µmol/L | µmol/L | µmol/L | µmol/L |
Chromatography:
| Chromatography ID: | CH001351 |
| Instrument Name: | Agilent 1290 Infinity |
| Column Name: | Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å) |
| Column Temperature: | 40 |
| Flow Gradient: | 20% B: 2 min, 20% - 60% B: 2 - 7 min, 60% - 100% B: 7 - 9 min, 20% B: 9 - 10.8 min |
| Flow Rate: | 0.4 mL/min |
| Sample Injection: | 2 µL |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001352 |
| Instrument Name: | Agilent 1290 Infinity |
| Column Name: | Agilent Zorbax RRHD Eclipse Plus C18 (50 x 2.1mm,1.8um,95 Å) |
| Column Temperature: | 40 |
| Flow Gradient: | 20% B: 2 min, 20% - 60% B: 2 - 7 min, 60% - 100% B: 7 - 9 min, 20% B: 9 - 10.8 min |
| Flow Rate: | 0.4 mL/min |
| Sample Injection: | 1 µL |
| Solvent A: | 60% acetonitrile/40% water; 10 mM ammonium formate |
| Solvent B: | 90% isopropanol/10% acetonitrile; 10 mM ammonium formate |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001353 |
| Instrument Name: | Agilent 1290 Infinity |
| Column Name: | Waters Acquity BEH HILIC (100 x 2.1mm,1.7um,130 Å) |
| Column Temperature: | 60 |
| Flow Gradient: | 99.9% B: 0 - 5 min; 40% B: 5 - 5.5 min; 10% B: 5.5 - 6.6 min and 99.9% B: 6.6 - 9.6 min (total run time 9.6 min) |
| Flow Rate: | 0.4 mL/min |
| Sample Injection: | 2 µL |
| Solvent A: | 50% water/50% acetonitrile; 25 mM ammonium formate, pH 4.6 |
| Solvent B: | 95% acetonitrile/5% water; 25 mM ammonium formate, pH 4.6 |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH001354 |
| Instrument Name: | Agilent 1100 |
| Column Name: | Agilent Zorbax Eclipse XDB C18 (150 x 3.0mm,1.8um) |
| Column Temperature: | 25 |
| Flow Gradient: | Isocratic (25 min) |
| Flow Rate: | 0.128 mL/min |
| Sample Injection: | 10 µL |
| Solvent A: | 50% chloroform/50% methanol; 2 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS001726 |
| Analysis ID: | AN001870 |
| Instrument Name: | Agilent 6460 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Phospholipids, cholesteryl esters and diacylglycerols were measured with an Agilent 6460 triple quadrupole mass spectrometer in dynamic MRM mode. The ESI source settings were: polarity: positive, dry gas temperature 300°C, dry gas flow 5L/min, nebulizer pressure: 45 psi, sheath gas temperature: 250°C, sheath gas flow: 11 L/min, capillary voltage: 3500 V, noozle: 500. MRM transitions with collision energies are detailed in the attached protocol. Data were processed with Agilent MassHunter QQQ Quantitative Analysis (version B.08). For plasmalogen PE (PE-P), transitions with the fatty acid as product were used as quantifiers, and those with the head group as qualifiers. Plasmalogen PCs (PC-P), ether PCs (PC-O) and odd-chain fatty acid PCs were distinguished based on retention time (Alshehry et al., Circulation, 2016; Huynh et al, Cell Chem. Biol. 2019). Normalised peak areas were calculated by dividing the peak areas of the analyte with the corresponding class-specific internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the corresponding internal standard. Lipid species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation. |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 3500 V |
| Collision Gas: | Nitrogen |
| Dry Gas Flow: | 5 L/min |
| Dry Gas Temp: | 300°C |
| Fragment Voltage: | 135 V |
| Nebulizer: | 45 psi |
| MS ID: | MS001727 |
| Analysis ID: | AN001871 |
| Instrument Name: | Agilent 6495 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Sphingolipids were measured with an Agilent 6495A triple quadrupole mass spectrometer in dynamic MRM mode. The ESI source settings were: polarity: positive, dry gas temperature 200°C, dry gas flow 12 L/min, nebulizer pressure: 25 psi, sheath gas temperature: 250°C, sheath gas flow: 12 L/min, capillary voltage: 3500 V, noozle: 500, iFunnel high pressure RF: 80, iFunnel high pressure RF: 40. MRM transitions with collision energies are detailed in the attached protocol. Data were processed with Agilent MassHunter QQQ Quantitative Analysis (version B.08). Transitions of precursor ions with water loss were used as qualifiers. Normalised peak areas were calculated by dividing the peak areas of the analyte with the corresponding class-specific internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the corresponding internal standard. Lipid species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation. |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 3500 V |
| Collision Gas: | Nitrogen |
| Dry Gas Flow: | 12 L/min |
| Dry Gas Temp: | 200°C |
| Fragment Voltage: | 135 V |
| Nebulizer: | 25 psi |
| MS ID: | MS001728 |
| Analysis ID: | AN001872 |
| Instrument Name: | Agilent 6490 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Derivatized S1P species were measured with an Agilent 6490 triple quadrupole mass spectrometer in MRM mode. The ESI source settings were: polarity: positive, dry gas temperature 200°C, dry gas flow 12 L/min, nebulizer pressure: 25 psi, sheath gas temperature: 400°C, sheath gas flow: 12 L/min, capillary voltage: 3500 V, noozle: 500, iFunnel high pressure RF: 200, iFunnel high pressure RF: 110. MRM transitions with collision energies are detailed in the attached protocol. Data were processed with Agilent MassHunter QQQ Quantitative Analysis (version B.08). The m/z 60 fragments were used as quantifiers, the m/z 103 fragments as qualifiers. Normalised peak areas were calculated by dividing the peak areas of the S1P species with the internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the corresponding internal standard. S1P species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation. |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 3500 V |
| Collision Gas: | Nitrogen |
| Dry Gas Flow: | 12 L/min |
| Dry Gas Temp: | 200°C |
| Fragment Voltage: | 135 V |
| Nebulizer: | 25 psi |
| MS ID: | MS001729 |
| Analysis ID: | AN001873 |
| Instrument Name: | ABI Sciex 4000 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Triacylglycerol species were measured with a Sciex 4000 QTRAP mass spectrometer operated in single-ion monitoring (SIM) mode at unit resolution. The ESI source settings were: polarity: positive, electrospray voltage: 5 kV, source temperature: 250°C, drying gas: nitrogen, gas 1 flow: 40 units, gas 2 flow: 30 units and curtain gas flow: 10 units. MRM transitions with collision energies are detailed in the attached protocol. Raw data were processed with Sciex Analyst (Version 1.6.2). Normalised peak areas were calculated by dividing the peak areas of the TG species with the internal standard. Relative abundance was obtained by multiplying the normalised peak areas with the molar concentration of the internal standard. Lipid species with a median peak area in the PQC samples below 250 or less than 5 times of the highest signal in Blank samples were excluded. Additionally, the coefficient of variation (CV) of the normalised peak area was calculated for each lipid species in the PQC samples of each experimental group. Species with a CV higher than 25% in any of the two groups were excluded from subsequent evaluation. |
| Ion Mode: | POSITIVE |
| Ion Spray Voltage: | 5 kV |
| Source Temperature: | 250°C |
