Summary of Study ST001036

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 PR000693. The data can be accessed directly via it's Project DOI: 10.21228/M83H4H This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST001036
Study Title	TCA Concentrations in Neuromyelitis Optica Patients (part - II)
Study Summary	Patients with an inflammatory disease of the central nervous system known as neuromyelitis optica (NMO) experience increased levels of depression. These patients have an antibody that recognizes a type of cell in the brain called astrocytes – binding of this antibody to astrocytes triggers a stress response in the cell that results in the development of brain lesions that cause disability and cognitive disturbances. We recently observed a change in the level of glutamate in a part of the brain involved in depression in patients with NMO. Glutamate is a chemical that is used in the brain for communication between neurons – reduced levels of glutamate are thought to trigger depression by reducing neuronal activity in specific circuits. Based on this observation and the known role of astrocytes in maintaining glutamate levels in the brain, we hypothesize that the NMO antibody disturbs metabolic activity in astrocytes and thereby reduces glutamate and triggers depression. We intend to measure TCA concentration in NMO astrocytes. It is our hope that we will not only learn something about the mechanisms of astrocyte dysregulation in neuromyelitis optica, but that we will learn something about the mechanisms of depression in general that may lead to new therapies for this disease.
Institute	Mayo Clinic
Last Name	Howe
First Name	Charles
Address	200 First St. SW, Rochester, Minnesota, 55905, USA
Email	howe@mayo.edu
Phone	507-284-9288
Submit Date	2018-08-13
Analysis Type Detail	GC-MS
Release Date	2020-08-20
Release Version	1

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Project:

Project ID:	PR000693
Project DOI:	doi: 10.21228/M83H4H
Project Title:	Mayo Pilot and Feasibility: The pathogenic NMO IgG dysregulates the astrocytic glutamine-glutamate cycle: a metabolic basis for depression in NMO patients
Project Summary:	Neuromyelitis optica (NMO) is a disabling central nervous system (CNS) inflammatory disorder that involves a pathogenic autoantibody (NMO IgG) directed against aquaporin-4, the major brain water channel, expressed on astrocytes. Astrocytes in brain tissue from patients with NMO exhibit a spectrum of abnormalities and pathologies ranging from sublytic gliosis and reactivity to outright destruction. Our current working model for NMO pathogenesis involves an early and robust NMO IgG-induced astrocytic stress response that drives metabolic dyshomeostasis and the production of pro-inflammatory cytokines and chemokines that amplify pathology by recruiting innate immune effector cells to the CNS. Notably, astrocytes are implicated in clinical depression and patients with NMO experience depression at levels that exceed the general population. Preliminary magnetic resonance spectroscopy data from our group indicates that glutamate levels are reduced in the prefrontal cortex of NMO patients, suggesting that unipolar depression in these individuals is a direct pathogenic effect of NMO IgG-induced astrocytic dysregulation. Because astrocytes are critical for glutamine-glutamate cycling in the CNS, we hypothesize that stimulation of primary astrocytes with patient-derived NMO IgG will drive a metabolic shift marked by alterations in cellular levels of glutamate and glutamine. In preliminary experiments using 1H-NMR to measure metabolic changes induced in astrocytes by stimulation with NMO IgG we observed variable glutamate and glutamine responses. To overcome issues of signal-to-noise and the high basal levels of glutamate and glutamine produced by astrocytes, we now propose to use isotopic tracing and 13C-NMR to quantify NMO IgG-induced metabolic dysregulation. Our ultimate goal is to use NMO IgG-induced dyshomeostasis as a microscope to reveal basic mechanisms of pathogenic glutamate-glutamine metabolism in astrocytes that may not only impact the health of patients with NMO but may also yield novel insights into the mechanisms of depression in general.
Institute:	Mayo Clinic
Last Name:	Howe
First Name:	Charles
Address:	200 First St. SW, Rochester, Minnesota, 55905, USA
Email:	howe@mayo.edu
Phone:	507-284-9288

Subject:

Subject ID:	SU001075
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

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

mb_sample_id	local_sample_id	Time Point	Matrix	Condition
SA069331	ms6982-56	0h	Fresh Media	BGP 50uMGt Gn
SA069332	ms6923-21	0h	Fresh Media	BGP50uMGt Gn
SA069333	ms6923-19	0h	Fresh Media	BGPGt 0.5 mM Gn
SA069334	ms6982-54	0h	Fresh Media	BGPGt 0.5mM Gn
SA069335	ms6923-20	0h	Fresh Media	BGPGt 5mM Gn
SA069336	ms6982-55	0h	Fresh Media	BGPGt 5mM Gn
SA069337	ms6982-57	0h	Fresh Media	E Med
SA069338	ms6923-22	0h	Fresh Media	E Med
SA069339	ms6982-53	1h nonCO2	Spent Sup	BGP50uMGt Gn
SA069340	ms6923-18	1h nonCO2	Spent Sup	BGP50uMGt Gn
SA069341	ms6982-52	1h nonCO2	Spent Sup	BGP50uMGt Gn
SA069342	ms6982-51	1h nonCO2	Spent Sup	BGP50uMGt Gn
SA069343	ms6923-16	1h nonCO2	Spent Sup	BGP50uMGt Gn
SA069344	ms6923-17	1h nonCO2	Spent Sup	BGP50uMGt Gn
SA069345	ms6923-10	1h nonCO2	Spent Sup	BGPGt 0.5 mM Gn
SA069346	ms6923-12	1h nonCO2	Spent Sup	BGPGt 0.5 mM Gn
SA069347	ms6923-11	1h nonCO2	Spent Sup	BGPGt 0.5 mM Gn
SA069348	ms6982-46	1h nonCO2	Spent Sup	BGPGt 0.5mM Gn
SA069349	ms6982-45	1h nonCO2	Spent Sup	BGPGt 0.5mM Gn
SA069350	ms6982-47	1h nonCO2	Spent Sup	BGPGt 0.5mM Gn
SA069351	ms6982-50	1h nonCO2	Spent Sup	BGPGt 5mM Gn
SA069352	ms6982-48	1h nonCO2	Spent Sup	BGPGt 5mM Gn
SA069353	ms6923-13	1h nonCO2	Spent Sup	BGPGt 5mM Gn
SA069354	ms6982-49	1h nonCO2	Spent Sup	BGPGt 5mM Gn
SA069355	ms6923-14	1h nonCO2	Spent Sup	BGPGt 5mM Gn
SA069356	ms6923-15	1h nonCO2	Spent Sup	BGPGt 5mM Gn
SA069366	ms6982-43	23h +1h nonCO2	pellet	BGP50uMGt Gn
SA069367	ms6982-42	23h +1h nonCO2	pellet	BGP50uMGt Gn
SA069368	ms6982-44	23h +1h nonCO2	pellet	BGP50uMGt Gn
SA069357	ms6923-7	23h + 1h nonCO2	Pellet	BGP50uMGt Gn
SA069358	ms6923-8	23h + 1h nonCO2	Pellet	BGP50uMGt Gn
SA069359	ms6923-9	23h + 1h nonCO2	Pellet	BGP50uMGt Gn
SA069369	ms6982-36	23h +1h nonCO2	pellet	BGPGt 0.5mM Gn
SA069370	ms6982-38	23h +1h nonCO2	pellet	BGPGt 0.5mM Gn
SA069371	ms6982-37	23h +1h nonCO2	pellet	BGPGt 0.5mM Gn
SA069360	ms6923-3	23h + 1h nonCO2	Pellet	BGPGt 0.5 mM Gn
SA069361	ms6923-1	23h + 1h nonCO2	Pellet	BGPGt 0.5 mM Gn
SA069362	ms6923-2	23h + 1h nonCO2	Pellet	BGPGt 0.5 mM Gn
SA069372	ms6982-41	23h +1h nonCO2	pellet	BGPGt 5mM Gn
SA069373	ms6982-39	23h +1h nonCO2	pellet	BGPGt 5mM Gn
SA069374	ms6982-40	23h +1h nonCO2	pellet	BGPGt 5mM Gn
SA069363	ms6923-4	23h + 1h nonCO2	Pellet	BGPGt 5mM Gn
SA069364	ms6923-5	23h + 1h nonCO2	Pellet	BGPGt 5mM Gn
SA069365	ms6923-6	23h + 1h nonCO2	Pellet	BGPGt 5mM Gn
SA069375	ms6982-60	23h	Spent Sup	E Med
SA069376	ms6982-58	23h	Spent Sup	E Med
SA069377	ms6982-59	23h	Spent Sup	E Med
SA069378	ms6923-26	23h	Spent Sup	E Med
SA069379	ms6923-23	23h	Spent Sup	E Med
SA069380	ms6923-24	23h	Spent Sup	E Med
SA069381	ms6923-25	23h	Spent Sup	E Med

Showing results 1 to 51 of 51

Showing results 1 to 51 of 51

Collection:

Collection ID:	CO001069
Collection Summary:	4,000,000 CLOD cells collected by methanol scraping. 1ml sup also collected. There was no tracer element added to the sample. Also methanol extarction was done in ice. After the extraction samples were kept in dry ice and delivered to core.
Sample Type:	astrocytes

Treatment:

Treatment ID:	TR001089
Treatment Summary:	Murine derived pure cortical astrocytes were plated at 4x10^6 density in 10cm petri dish and grown in astro growth medium (AGM) for 2-3 days until it reaches the 90% confluency. Day before the treatment with glutamine or glutamate AGM was changed to serum free “E” media. In case of isotomer analysis 40% of glucose was switched with U13C6 (glucose) and added to serum free “E” media. On the day of treatment first the spent media was collected and then washed with base media. After washing, astrocytes were exposed to different ratio of glutamate: glutamine for 1h under non-CO2 condition. 1h spent media was collected. After washing three times with phosphate buffer saline cell pellet was collected in ice cold 100% methanol by scrapping. All the samples were immediately transferred to -70C for future analysis. E media composition: DMEM (w/o phenol red)+ 1% PenStrep+ 10% FBS+ Glutamate+ Glucose Abbreviation: Gn-Glutamine Gt-Glutamate B- Base media G- Glucose P- Sodium pyruvate

Treatment ID:

TR001089

Treatment Summary:

Murine derived pure cortical astrocytes were plated at 4x10^6 density in 10cm petri dish and grown in astro growth medium (AGM) for 2-3 days until it reaches the 90% confluency. Day before the treatment with glutamine or glutamate AGM was changed to serum free “E” media. In case of isotomer analysis 40% of glucose was switched with U13C6 (glucose) and added to serum free “E” media. On the day of treatment first the spent media was collected and then washed with base media. After washing, astrocytes were exposed to different ratio of glutamate: glutamine for 1h under non-CO2 condition. 1h spent media was collected. After washing three times with phosphate buffer saline cell pellet was collected in ice cold 100% methanol by scrapping. All the samples were immediately transferred to -70C for future analysis. E media composition: DMEM (w/o phenol red)+ 1% PenStrep+ 10% FBS+ Glutamate+ Glucose Abbreviation: Gn-Glutamine Gt-Glutamate B- Base media G- Glucose P- Sodium pyruvate

Sample Preparation:

Sampleprep ID:	SP001082
Sampleprep Summary:	TCA concentrations of Astrocytes We intend to test the experimental hypothesis that stimulation of primary murine astrocytes with patient-derived NMO IgG will drive a metabolic shift marked by alterations in cellular levels of glutamate and glutamine. To do so we will use isotopic tracing to measure glutamate and glutamine levels in cell extracts following stimulation with NMO IgG. The cell preparations, patient-derived antibody preps, isotopic tracer incubations, and cell stimulations will be performed in the PI’s lab.

Sampleprep ID:

SP001082

Sampleprep Summary:

TCA concentrations of Astrocytes We intend to test the experimental hypothesis that stimulation of primary murine astrocytes with patient-derived NMO IgG will drive a metabolic shift marked by alterations in cellular levels of glutamate and glutamine. To do so we will use isotopic tracing to measure glutamate and glutamine levels in cell extracts following stimulation with NMO IgG. The cell preparations, patient-derived antibody preps, isotopic tracer incubations, and cell stimulations will be performed in the PI’s lab.

Combined analysis:

Analysis ID	AN001697
Analysis type	MS
Chromatography type	GC
Chromatography system	Agilent 7890B
Column	Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type	EI
MS instrument type	Single quadrupole
MS instrument name	Agilent 5977A
Ion Mode	POSITIVE
Units	nmol/vial

Chromatography:

Chromatography ID:	CH001196
Instrument Name:	Agilent 7890B
Column Name:	Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Chromatography Type:	GC

MS:

MS ID:	MS001572
Analysis ID:	AN001697
Instrument Name:	Agilent 5977A
Instrument Type:	Single quadrupole
MS Type:	EI
Ion Mode:	POSITIVE