Summary of Study ST001864

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 PR001177. The data can be accessed directly via it's Project DOI: 10.21228/M8K41X This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001864
Study TitleTargeting host glycolysis as a strategy for antimalarial development
Study SummaryGlycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.
Institute
University of Colorado Anschutz Medical Campus
Last NameHaines
First NameJulie
Address12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Emailjulie.haines@cuanschutz.edu
Phone3037243339
Submit Date2021-07-02
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2021-07-24
Release Version1
Julie Haines Julie Haines
https://dx.doi.org/10.21228/M8K41X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001177
Project DOI:doi: 10.21228/M8K41X
Project Title:Targeting host glycolysis as a strategy for antimalarial development
Project Summary:Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase – a critical enzyme in glycolysis – and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.
Institute:University of Colorado Anschutz Medical Campus
Last Name:Haines
First Name:Julie
Address:12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Email:julie.haines@cuanschutz.edu
Phone:3037243339

Subject:

Subject ID:SU001941
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id group
SA17444234Control, 0.5h
SA17444332Control, 0.5h
SA17444436Control, 0.5h
SA1744452Control, 0h
SA1744466Control, 0h
SA1744474Control, 0h
SA17444866Control, 1h
SA17444964Control, 1h
SA17445062Control, 1h
SA174451124Control, 6h
SA174452126Control, 6h
SA174453122Control, 6h
SA17445456POMHEX, 0.5h
SA17445560POMHEX, 0.5h
SA17445658POMHEX, 0.5h
SA17445730POMHEX, 0h
SA17445826POMHEX, 0h
SA17445928POMHEX, 0h
SA17446088POMHEX, 1h
SA17446190POMHEX, 1h
SA17446286POMHEX, 1h
SA174463150POMHEX, 6H
SA174464148POMHEX, 6H
SA174465146POMHEX, 6H
SA17446652POMSF, 0.5h
SA17446754POMSF, 0.5h
SA17446850POMSF, 0.5h
SA17446922POMSF, 0h
SA17447024POMSF, 0h
SA17447120POMSF, 0h
SA17447284POMSF, 1h
SA17447380POMSF, 1h
SA17447482POMSF, 1h
SA174475140POMSF, 6H
SA174476142POMSF, 6H
SA174477144POMSF, 6H
Showing results 1 to 36 of 36

Collection:

Collection ID:CO001934
Collection Summary:Banked blood (Saint Louis Children’s Hospital) and fresh blood [healthy donors] was washed and stored at 50% hematocrit at 4ºC up to 1 month past the clinical expiration date and collection date, respectively. Parasite strains provided by the Malaria Research and Reference Reagent Resource Center (MR4) as follows: 3D7 (MRA-102); K1 (MRA-159); D10 (MRA-201); and IPC-5202 (MRA-1240). Unless indicated, erythrocytes were cultured as previously described at 2% hematocrit in complete media (incomplete media supplemented with 27mM sodium bicarbonate, 11mM glucose, 5mM HEPES, 1mM sodium pyruvate, 0.37mM hypoxanthine, 0.01mM thymidine, 10ug/mL gentamicin, and 0.5% Albumax (Thermo-Fisher) under 5% O2/5% CO2/90% N2 atmosphere at 37ºC (71).
Sample Type:Blood (whole)

Treatment:

Treatment ID:TR001953
Treatment Summary:Freshly collected erythrocytes were washed, resuspended to 10% hematocrit in buffer [25mM HEPES (pH 7.4), 120mM NaCl, 5.4mM KCl, 1.8mM CaCl2, and 1mM NaH2PO4], incubated for 1 hour at 37ºC, then centrifuged at 2000xg for 5 minutes at 4ºC, resuspended in fresh wash buffer to 30-40% hematocrit, and split into 210uL aliquots of packed erythrocytes. Erythrocytes were resuspended in 253uL of RPMI containing 11.9mM D-[1,2,3-13C] glucose (Millipore-Sigma) and either treated with POM-SF or POM-HEX at five times the EC50s for MetHb formation, or an untreated solvent control with the balance volume to 550uL using washing buffer. All samples were incubated at 37ºC shaking at 500 RPM. Triplicate samples of supernatants and packed erythrocytes were collected at 0, 0.5, 1, and 6-hour intervals and snap-frozen in liquid N2.

Sample Preparation:

Sampleprep ID:SP001947
Sampleprep Summary:RBC and media samples were extracted at 1:10 and 1:20 dilutions, respectively, in ice-cold 5:3:2 MeOH:MeCN:H2O v/v/v, with vigorous vortexing at 4 degrees C for 30 min. The extraction buffer was supplemented with 40uM 3-13C lactate to allow absolute quantification of lactate isotopologues, including lactate generated via glycolysis ([1,2,3-13C3] enriched) vs. the pentose phosphate pathway ([2,3-13C2] enriched). Supernatants were clarified via centrifugation (10,000 g, 10 min, 4 C) then analyzed by UHPLC-MS.

Combined analysis:

Analysis ID AN003021 AN003022
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Vanquish Vanquish
Column Kinetex XB-C18 (150 x 2.1mm,1.7um) Kinetex XB-C18 (150 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE POSITIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH002239
Chromatography Summary:10 μl of RBC extracts were injected and separated through a 3 min isocratic elution on a Kinetex XB-C18 column (150 × 2.1 mm i.d., 1.7 μm particle size – Phenomenex, Torrance, CA, USA) at 250 μl/min (mobile phase: 5% acetonitrile, 95% 18 mΩ H2O, 0.1% formic acid; column temperature: 25°C).
Instrument Name:Vanquish
Column Name:Kinetex XB-C18 (150 x 2.1mm,1.7um)
Column Temperature:25
Flow Gradient:isocratic
Flow Rate:250 ul/min
Solvent A:5% acetonitrile/95%water; 0.1% formic acid
Solvent B:isocratic
Chromatography Type:Reversed phase

MS:

MS ID:MS002810
Analysis ID:AN003021
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs. MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above.
Ion Mode:NEGATIVE
  
MS ID:MS002811
Analysis ID:AN003022
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The UHPLC system was coupled online with a QExactive mass spectrometer (Thermo, San Jose, CA, USA), scanning in Full MS mode (2 μscans) at 70,000 resolution from 60-900 m/z, with 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in positive ion mode. Calibration was performed before each analysis using a positive calibration mix (Piercenet – Thermo Fisher, Rockford, IL, USA). Limits of detection (LOD) were characterized by determining the smallest injected amino acid amount required to provide a signal to noise (S/N) ratio greater than three using < 5 ppm error on the accurate intact mass. Based on a conservative definition for Limit of Quantitation (LOQ), these values were calculated to be three fold higher than determined LODs. MS data acquired from the QExactive was converted from .raw file format to.mzXML format using MassMatrix (Cleveland, OH, USA). Amino acid assignments were performed using MAVEN (Princeton, NJ, USA). The MAVEN software platform provides tools for peak picking, feature detection and metabolite assignment against the KEGG pathway database. Assignments were further confirmed using a process for chemical formula determination using isotopic patterns and accurate intact mass (Clasquin et al. 2012). Analyte retention times were confirmed by comparison with external standard retention times, as indicated above.
Ion Mode:POSITIVE
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