Summary of study ST001052

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000705. The data can be accessed directly via it's Project DOI: 10.21228/M8JH5X This work is supported by NIH grant, U2C- DK119886.

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Study IDST001052
Study TitleLipidomics for wildlife disease etiology and biomarker discovery: a case study of pansteatitis outbreak in South Africa (part-I)
Study Typelipidomics
Study SummaryLipidomics is a promising tool to determine biomarkers and elucidate mechanisms associated with anthropogenic-induced stress in wildlife. Therefore, we examine the application of lipidomics for in situ studies on Mozambique tilapia (Oreochromis mossambicus) in Loskop Dam, South Africa. Mortality events of aquatic life associated with an environmentally-derived inflammatory disease, pansteatitis, have occurred in this area. The lipidome of adipose tissue (n = 31) and plasma (n = 51) from tilapia collected from at Loskop Dam were characterized using state of the art liquid chromatography coupled to high-resolution tandem mass spectrometry. Lipid profiles reflected pansteatitis severity and were significantly different between diseased and healthy individuals. Over 13 classes of lipids associated with inflammation, cell death, and/or oxidative damage were upregulated in pansteatitis-affected adipose tissue, including ether-lipids, short-chained triglyceride oxidation products, sphingolipids, and acylcarnitines. Ceramides showed a 1000-fold increase in the most affected adipose tissues, illustrating its potential as sensitive and novel indicators of disease severity. In plasma, triglycerides were found to be downregulated in pansteatitis-affected tilapia. As comprehensive coverage of the lipidome aids in the elucidation of possible disease mechanisms, application of lipidomics could be applied to the understanding of other environmentally-derived inflammatory conditions, such as those caused by obesogens.
Institute
South East Center for Integrated Metabolomics
DepartmentDepartment of Pathology, Immunology and Laboratory Medicine
LaboratorySECIM
Last NameKoelmel
First NameJeremy
AddressDepartment of Pathology, Immunology and Laboratory Medicine, University of Florida, 1395 Center Dr, Room M641c
Emailjeremykoelmel@gmail.com
Phone7187300454
Submit Date2018-09-13
Num Groups8
Total Subjects51
Num Males28
Num Females23
Study CommentsAdipose and plasma do not overlap exactly in subjects ran
Publicationssubmitted to Metabolomics
Raw Data AvailableYes
Analysis Type DetailLC-MS
Release Date2018-09-27
Release Version1
Jeremy Koelmel Jeremy Koelmel
https://dx.doi.org/10.21228/M8JH5X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000705
Project DOI:doi: 10.21228/M8JH5X
Project Title:Lipidomics for wildlife disease etiology and biomarker discovery: a case study of pansteatitis outbreak in South Africa
Project Type:Lipidomics
Project Summary:The lipidome of adipose tissue (n = 31) and plasma (n = 51) from Tilapia collected at Loskop Dam, South Africa, were characterized using state of the art liquid chromatography coupled to high-resolution tandem mass spectrometry. Lipid profiles reflected pansteatitis severity and were significantly different between diseased and healthy individuals. Over 13 classes of lipids associated with inflammation, cell death, and/or oxidative damage were upregulated in pansteatitis-affected adipose tissue, including ether-lipids, short-chained triglyceride oxidation products, sphingolipids, and acylcarnitines. Ceramides showed a 1000-fold increase in the most affected adipose tissues, illustrating its potential as sensitive and novel indicators of disease severity. In plasma, triglycerides were found to be downregulated in pansteatitis-affected tilapia. As comprehensive coverage of the lipidome aids in the elucidation of possible disease mechanisms, application of lipidomics could be applied to the understanding of other environmentally-derived inflammatory conditions, such as those caused by obesogens.
Institute:South East Center for Integrated Metabolomics
Department:Department of Pathology, Immunology and Laboratory Medicine
Laboratory:SECIM
Last Name:Koelmel
First Name:Jeremy
Address:Department of Pathology, Immunology and Laboratory Medicine, University of Florida, 1395 Center Dr, Room M641c, Gainesville, FL, 32610, USA
Email:jeremykoelmel@gmail.com
Phone:7187300454
Funding Source:NIH Grant #U24 DK097209
Publications:submitted to Metabolomics
Contributors:Jeremy P. Koelmel, Candice Z. Ulmer, Susan Fogelson, Christina M. Jones, Hannes Botha, Jacqueline T. Bangma, Theresa C. Guillette, Wilmien J. Luus-Powell, Joseph R. Sara, Willem J. Smit, Korin Albert, Harmony A. Miller, Matthew P. Guillette, Berkley C. Olsen, Jason A. Cochran, Timothy J. Garrett, Richard A. Yost, John A. Bowden

Subject:

Subject ID:SU001094
Subject Type:Fish
Subject Species:Oreochromis mossambicus
Taxonomy ID:8127
Age Or Age Range:1 to 14 years
Weight Or Weight Range:0.1 to 5.6 kg
Height Or Height Range:NA
Gender:Male and female

Factors:

Subject type: Fish; Subject species: Oreochromis mossambicus (Factor headings shown in green)

mb_sample_id local_sample_id Group Gender
SA0714118399FD F
SA0714128393FD F
SA0714138403FD F
SA0714148358FD F
SA0714158392FD F
SA0714168404FD F
SA0714178390FD F
SA0714188371FD F
SA0714198370FD F
SA0714208363FD F
SA0714218373FD F
SA0714228376FD F
SA0714238385FD F
SA0714248379FD F
SA0714258378FD F
SA0714268407FH F
SA0714278405FH F
SA0714288356FH F
SA0714298396FH F
SA0714308382FH F
SA0714318387FH F
SA0714328391FH F
SA0714338394FH F
SA071409Maybe affected- -
SA071410hard to tell with empty granulomas"- -
SA0714348395MD M
SA0714358384MD M
SA0714368397MD M
SA0714378383MD M
SA0714388402MD M
SA0714398400MD M
SA0714408368MD M
SA0714418367MD M
SA0714428360MD M
SA0714438380MD M
SA0714448372MD M
SA0714458359MD M
SA0714468375MD M
SA0714478377MD M
SA0714488374MD M
SA0714498406MH M
SA0714508401MH M
SA0714518408MH M
SA0714528410MH M
SA0714538398MH M
SA0714548409MH M
SA0714558366MH M
SA0714568362MH M
SA0714578355MH M
SA0714588364MH M
SA0714598381MH M
SA0714608386MH M
SA0714618389MH M
Showing results 1 to 53 of 53

Collection:

Collection ID:CO001088
Collection Summary:Sample Collection and Metadata Fifty one (n=51) Mozambique tilapia (Oreochromis mossambicus) were captured at various locations along the inflow of Loskop Dam, Mpumalanga, South Africa (25°28'22.8" S: 29°15'25.2" E) between May 2-6, 2016. On capture, blood was immediately drawn and collected in lithium heparin 3 mL vacutainers (BD vacutainer, Franklin Lakes, NJ). After the first blood draw, fish were kept in aerated tanks and transported back to the field station for necropsy. All fish work was conducted using the permit and animal handling protocol that was reviewed and approved by the Mpumalanga Tourism and Parks Agency (Project #ES 6/1 ). Upon arrival to the field station, blood was immediately centrifuged in collection tubes and 500 μL of plasma was aliquoted into cryogenic storage vials (Corning, Corning, NY) for lipidomic studies (n=51). Adipose tissue was removed during dissection and flash frozen and stored for lipidomics analysis. Juvenile fish with an ambiguous sex assignment were excluded. Pictures of the whole fish and individual biopsied tissues were taken under consistent lighting in a photo box for documentation. A subset of the sampled fish, representing individuals with varying degrees of disease severity with matched age, size, and weight, were selected for lipidomics of adipose tissue (n=31).
Sample Type:Blood (plasma)
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001108
Treatment Summary:Mozambique tilapia were not treated, but rather captured in the wild.

Sample Preparation:

Sampleprep ID:SP001101
Sampleprep Summary:Sample Preparation for Liquid Chromatography - Mass Spectrometric Analysis Lipid internal standards were purchased from the following locations to generate a lipid internal standard mix: Nu-Chek Prep (CE 19:0, DG 19:2/19:2, DG 20:0/20:0, MG 17:0, TG 13:0/13:0/13:0, TG 17:1/17:1/17:1, and TG 19:0/19:0/19:0), and Avanti Polar Lipids (Cer d18:1/25:0, free cholesterol-d7, GalCer d18:1/12:0, GlcCer d18:1/12:0, LPA 17:0, LPC 19:0, LPE 14:0, LSM d17:1, OxPC 16:0/9:0, PA 14:0/14:0, PC 14:1/14:1, PC 19:0/19:0, PE 15:0/15:0, PE 17:0/17:0, PG 15:0/15:0, PG 17:0/17:0, PI 8:0/8:0, PS 14:0/14:0, and SM d18:1/6:0). Optima methanol and HPLC grade chloroform were purchased from Thermo Fisher Scientific (Waltham, MA, USA). HPLC grade 2-propanol was acquired from Alfa Aesar (Haverhill, MA, USA). Ultrapure water with 18 MΩ cm resistivity (Millipore Milli-Q Gradient A10; EMD Millipore, Billerica, MA, USA) was used for sample preparation. Ammonium acetate and analytical grade formic acid were purchased from Fisher Scientific. All mobile phase solvents were Fisher Optima LC/MS grade (acetonitrile, isopropanol, and water). Plasma samples (50 μL) were extracted using the Bligh–Dyer extraction protocol.1 Briefly, samples were removed from the −80 °C freezer and thawed on ice. To each sample, a 4 mL chloroform:methanol mixture (1:1, v:v) was added followed by a 1.75 mL water addition, accounting for the water present in plasma. Samples were then incubated on ice for 30 min, vortexed for 20 s, and centrifuged at 2000 rpm for 10 min to separate the organic and aqueous layers. The organic layer was collected and the aqueous layer was re-extracted with 2 mL 1:1 (v:v, chloroform:methanol). The organic layers were combined, evaporated under nitrogen, and reconstituted in 50 μL of isopropanol for a 5 μL injection. The final average concentration of internal standards in mg standard per kg plasma wet weights were: CE(19:0) (1.06 mg/kg), Cer(d18:1/25:0) (116.99 mg/kg), DG(19:2/19:2) (0.08 mg/kg), DG(20:0/20:0) (0.73 mg/kg), GlcCer(d18:1/12:0) (0.13 mg/kg), LPA(17:0) (0.35 mg/kg), LPC(19:0) (2.48 mg/kg), LPE(14:0) (1.49 mg/kg), PA(14:0/14:0) (8.81 mg/kg), PC(14:1/14:1) (2.72 mg/kg), PC(19:0/19:0) (3.53 mg/kg), PE(15:0/15:0) (1.16 mg/kg), PE(17:0/17:0) (3.09 mg/kg), PG(15:0/15:0) (1.25 mg/kg), PG(17:0/17:0) (2.52 mg/kg), PS(14:0/14:0) (1.84 mg/kg), SM(d18:1/6:0) (0.15 mg/kg), TG(17:1/17:1/17:1) (1.53 mg/kg), and TG(19:0/19:0/19:0) (1.22 mg/kg). Whole adipose tissues were cryopulverized using a Retsch Cryomill (Retsch, Haan, Germany) and 30 mg aliquots of powder were removed for extraction. All powdered aliquots were extracted using the Folch method (2:1, v/v chloroform:methanol).2 Briefly, 100 μL of the internal standard mix was spiked into tissue samples on ice. Chloroform (2 mL) and methanol (1 mL) were added to all samples. Samples were vortexed and incubated on ice for 30 minutes. Upon the addition of water (750 μL) to induce phase separation, samples were vortexed and incubated on ice for an additional 10 min. Samples were centrifuged for 10 min at 2000 rpm. The organic layer was removed and the aqueous layer was re-extracted with 1500 µL of chloroform:methanol (2:1, v/v). The organic layers were combined, evaporated under nitrogen, and reconstituted in 2000 µL of isopropanol for a 2 μL injection. The final average concentrations of internal standards in adipose tissues were: CE(19:0) (71.40 mg/kg), Cer(d18:1/25:0) (7875.37 mg/kg), DG(19:2/19:2) (5.71 mg/kg), DG(20:0/20:0) (48.81 mg/kg), GalCer(d18:1/12:0) (8.86 mg/kg), GlcCer(d18:1/12:0) (8.87 mg/kg), LPA(17:0) (23.32 mg/kg), LPC(19:0) (167.27 mg/kg), LPE(14:0) (100.23 mg/kg), MG(17:0) (15.92 mg/kg), PA(14:0/14:0) (593.08 mg/kg), PC(14:1/14:1) (183.25 mg/kg), PC(19:0/19:0) (237.29 mg/kg), PE(15:0/15:0) (78.05 mg/kg), PE(17:0/17:0) (208.09 mg/kg), PG(15:0/15:0) (84.15 mg/kg), PG(17:0/17:0) (169.34 mg/kg), PI(8:0/8:0) (1.21 mg/kg), PS(14:0/14:0) (123.92 mg/kg), SM(d18:1/6:0) (9.91 mg/kg), TG(13:0/13:0/13:0) (86.90 mg/kg), TG(17:1/17:1/17:1) (103.22 mg/kg), and TG(19:0/19:0/19:0) (82.01 mg/kg). Data Acquisition All lipid extracts were randomized and analyzed by ultra-high performance liquid chromatography coupled to a high-resolution mass spectrometer (UHPLC-HRMS). Mass spectra were acquired on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with a heated electrospray ionization (HESI II) probe in positive and negative ion mode. HESI and mass spectrometric parameters for lipid extracts were as follows in positive/negative ion mode, respectively: spray voltage: 3.5/2.5 kV, sheath gas: 40/35 arbitrary units, auxiliary nitrogen pressure: 15 arbitrary units, sweep gas: 1/0 arbitrary units, ion transfer tube and vaporizer temperatures: 325 and 300/275 °C, and RF lens level: 30. Full scan, data-dependent MS/MS (ddMS2-top10), and data-independent acquisition mode (specifically all-ion fragmentation (AIF)) data were collected at m/z 150–2000, corresponding to the mass range of most expected cellular lipids. For ddMS2-top10, iterative exclusion,3 where lipids selected for fragmentation are excluded from fragmentation in a sequential injection, was applied. This method drastically improves lipid coverage.3 External calibration was applied before each run to allow for full scan LC-HRMS analysis at 120,000 resolution (m/z 200 FWHM). A Thermo Scientific Vanquish UHPLC system (Thermo Scientific, San Jose, CA) was coupled to the Orbitrap Fusion Lumos Tribrid for the chromatographic separation of lipids. The autosampler temperature was maintained at 4 °C for all experiments. Solvent extraction blanks and quality control samples were jointly analyzed over the course of a batch (10–15 samples). A Waters Acquity C18 BEH column (2.1 × 100 mm, 1.7 μm particle size, Waters, Milford, MA) maintained at 60 °C was used for all lipidomic studies. The mobile phase flow rate was 450 μL/min. The gradient program consisted of mobile phase C [60:40 acetonitrile/water] and mobile phase D [90:8:2 isopropanol/ acetonitrile/water], each containing 10 mM ammonium formate and 0.1% formic acid. The gradient included 32% D at 0 min, 40% D at 1 min, a hold at 40% D until 1.5 min, 45% D at 4 min, 50% D at 5 min, 60% D at 8 min, 70% D at 11 min, 80% D at 14 min, 100% D at 16 min, and a hold at 100% D until 17 min. The total run time was 22 min, which included a 5-min equilibration. (1) Bligh, E. G.; Dyer, W. J. A Rapid Method of Total Lipid Extraction and Purification. Can. J. Biochem. Physiol. 1959, 37 (8), 911–917. (2) Folch, J.; Lees, M.; Sloane Stanley, G. H. A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues. J. Biol. Chem. 1957, 226 (1), 497–509. (3) Koelmel, J. P.; Kroeger, N. M.; Gill, E. L.; Ulmer, C. Z.; Bowden, J. A.; Patterson, R. E.; Yost, R. A.; Garrett, T. J. Expanding Lipidome Coverage Using LC-MS/MS Data-Dependent Acquisition with Automated Exclusion List Generation. J. Am. Soc. Mass Spectrom. 2017, 28 (5), 908–917.
Processing Storage Conditions:-80℃
Extraction Method:Folch

Combined analysis:

Analysis ID AN001721
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Waters Acquity BEH C18 (150 x 2.1mm, 1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Fusion Tribrid Orbitrap
Ion Mode UNSPECIFIED
Units mg/kg (relative quantification, NOT ABSOLUTE)

Chromatography:

Chromatography ID:CH001217
Chromatography Summary:A Thermo Scientific Vanquish UHPLC system (Thermo Scientific, San Jose, CA) was coupled to the Orbitrap Fusion Lumos Tribrid for the chromatographic separation of lipids. The autosampler temperature was maintained at 4 °C for all experiments. Solvent extraction blanks and quality control samples were jointly analyzed over the course of a batch (10–15 samples). A Waters Acquity C18 BEH column (2.1 × 100 mm, 1.7 μm particle size, Waters, Milford, MA) maintained at 60 °C was used for all lipidomic studies. The mobile phase flow rate was 450 μL/min. The gradient program consisted of mobile phase C [60:40 acetonitrile/water] and mobile phase D [90:8:2 isopropanol/ acetonitrile/water], each containing 10 mM ammonium formate and 0.1% formic acid. The gradient included 32% D at 0 min, 40% D at 1 min, a hold at 40% D until 1.5 min, 45% D at 4 min, 50% D at 5 min, 60% D at 8 min, 70% D at 11 min, 80% D at 14 min, 100% D at 16 min, and a hold at 100% D until 17 min. The total run time was 22 min, which included a 5-min equilibration.
Instrument Name:Thermo Vanquish
Column Name:Waters Acquity BEH C18 (150 x 2.1mm, 1.7um)
Column Temperature:60 °C
Flow Gradient:The gradient included 32% D at 0 min, 40% D at 1 min, a hold at 40% D until 1.5 min, 45% D at 4 min, 50% D at 5 min, 60% D at 8 min, 70% D at 11 min, 80% D at 14 min, 100% D at 16 min, and a hold at 100% D until 17 min. The total run time was 22 min, which included a 5-min equilibration.
Flow Rate:450 uL/min
Solvent A:60:40 acetonitrile/water 10 mM ammonium formate and 0.1% formic acid
Solvent B:90:8:2 isopropanol/ acetonitrile/water 10 mM ammonium formate and 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS001593
Analysis ID:AN001721
Instrument Name:Thermo Fusion Tribrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
Ion Mode:UNSPECIFIED
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