#METABOLOMICS WORKBENCH hormel101_20170712_151100 DATATRACK_ID:1126 STUDY_ID:ST000797 ANALYSIS_ID:AN001268 PROJECT_ID:PR000575
VERSION             	1
CREATED_ON             	July 13, 2017, 9:40 pm
#PROJECT
PR:PROJECT_TITLE                 	Mayo Pilot and Feasibility: Targeting Myelin Metabolism to Enhance Recovery of
PR:PROJECT_TITLE                 	Function after SCI
PR:PROJECT_SUMMARY               	The loss of myelin, degeneration of the myelin producing oligodendroglia and
PR:PROJECT_SUMMARY               	impaired remyelination are essential features of traumatic spinal cord injury
PR:PROJECT_SUMMARY               	(SCI) that significantly limit patient recovery of function. The lipid rich
PR:PROJECT_SUMMARY               	composition of myelin, including exceptionally high levels of saturated fatty
PR:PROJECT_SUMMARY               	acids, underlie its essential physiological roles, including its structural and
PR:PROJECT_SUMMARY               	signaling properties and electrical insulation of axons to facilitate the
PR:PROJECT_SUMMARY               	conduction of nerve impulses. The myelin sheaths also provide metabolic support
PR:PROJECT_SUMMARY               	to the axons they wrap, and myelin health is therefore essential to the
PR:PROJECT_SUMMARY               	maintenance of axon integrity and function in the brain and spinal cord. The
PR:PROJECT_SUMMARY               	primary goal of this Pilot Proposal to the Mayo Clinic Metabolomics Core is to
PR:PROJECT_SUMMARY               	integrate highly sensitive metabolomics liquid chromatography-tandem mass
PR:PROJECT_SUMMARY               	spectrometry (LC/MS/MS) assays to quantify the lipid composition of the myelin
PR:PROJECT_SUMMARY               	membrane, with our conventional neurobehavioral approaches, enabling us to
PR:PROJECT_SUMMARY               	explore the metabolic basis of new interventions capable of promoting myelin
PR:PROJECT_SUMMARY               	regeneration and restoration of patient function. Metabolomics Core expertise in
PR:PROJECT_SUMMARY               	Magnetic Resonance Spectroscopy (NMR) based evaluation of key metabolites
PR:PROJECT_SUMMARY               	involved in CNS injury and repair (N-acetyl-L-aspartate, choline, myo-inositol,
PR:PROJECT_SUMMARY               	glucose/ glutamine and lactate) will also be applied to strengthen our
PR:PROJECT_SUMMARY               	mechanistic understanding of myelin injury and repair. Specifically, utilizing
PR:PROJECT_SUMMARY               	these innovative approaches we will test a novel hypothesis driven by new
PR:PROJECT_SUMMARY               	preliminary findings that the levels of dietary fatty acids can be optimized
PR:PROJECT_SUMMARY               	alone, or in combination with exercise training, to facilitate myelin
PR:PROJECT_SUMMARY               	regeneration and recovery of neurobehavioral function after injury to the adult
PR:PROJECT_SUMMARY               	spinal cord. In Aim 1, we will determine whether alterations in dietary fat,
PR:PROJECT_SUMMARY               	including saturated fat or omega-3 fatty acids, facilitate restoration of the
PR:PROJECT_SUMMARY               	myelin membrane and metabolite signatures of central nervous system repair after
PR:PROJECT_SUMMARY               	experimental SCI in adult mice. In Aim 2, we will determine whether exercise
PR:PROJECT_SUMMARY               	training alone or in combination with dietary fatty acid supplementation fosters
PR:PROJECT_SUMMARY               	myelin regeneration and recovery of function after experimental SCI. The
PR:PROJECT_SUMMARY               	proposed studies will leverage the expertise of the Mayo Metabolomics Core with
PR:PROJECT_SUMMARY               	that of Dr. Scarisbrick (Mayo) in myelin biology and Dr. Gomez Pinilla (UCLA) in
PR:PROJECT_SUMMARY               	central nervous system plasticity to investigate whether two highly targetable
PR:PROJECT_SUMMARY               	lifestyle variables, that is diet and exercise, can be modulated to improve
PR:PROJECT_SUMMARY               	myelin metabolism and functional recovery after SCI.
PR:INSTITUTE                     	Mayo Clinic
PR:LAST_NAME                     	Scarisbrick
PR:FIRST_NAME                    	Isobel
PR:ADDRESS                       	200 First St. SW, Rochester, Minnesota, 55905, USA
PR:EMAIL                         	scarisbrick.isobel@mayo.edu
PR:PHONE                         	507-284-0124
#STUDY
ST:STUDY_TITLE                   	Targeted Galactosyl Sphingolipids Concentrations of Myelin to Enhance Recovery
ST:STUDY_TITLE                   	of Function after SCI
ST:STUDY_SUMMARY                 	Tissue is from adult mouse spinal cord (SC). We are submitting these samples for
ST:STUDY_SUMMARY                 	Untargeted Profiling (unbiased metabolomics assay) and for lipid analysis. The
ST:STUDY_SUMMARY                 	lipid assays we request are 1) free fatty acid composition of lipids; 2) free
ST:STUDY_SUMMARY                 	fatty acid panel; 3) cholesterol concentration (free and bound); 4) Ceramides,
ST:STUDY_SUMMARY                 	including galactosyl and glucosyl; 5) sphingomyelin. The Untargeted profiling is
ST:STUDY_SUMMARY                 	our top priority, followed by the lipid assays as listed. All samples were snap
ST:STUDY_SUMMARY                 	frozen at the point of harvest and approximate weights are provided. The samples
ST:STUDY_SUMMARY                 	are submitted as intact pieces of tissue. There are 20 samples total, n=5 for
ST:STUDY_SUMMARY                 	each group that includes LF (low fat diet); HF (high fat diet); HFHS (high fat
ST:STUDY_SUMMARY                 	high sucrose diet); and Keto (ketogenic diet).
ST:INSTITUTE                     	Mayo Clinic
ST:LAST_NAME                     	Scarisbrick
ST:FIRST_NAME                    	Isobel
ST:ADDRESS                       	200 First St. SW, Rochester, Minnesota, 55905, USA
ST:EMAIL                         	riska.shaun@mayo.edu
ST:PHONE                         	507-284-0124
#SUBJECT
SU:SUBJECT_TYPE                  	Mouse
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	ms6011-1	group:LFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-2	group:LFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-3	group:LFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-4	group:LFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-5	group:LFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-6	group:HFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-7	group:HFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-8	group:HFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-9	group:HFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-10	group:HFD	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-11	group:HFHS	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-12	group:HFHS	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-13	group:HFHS	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-14	group:HFHS	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-15	group:HFHS	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-16	group:Keto	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-17	group:Keto	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-18	group:Keto	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-19	group:Keto	
SUBJECT_SAMPLE_FACTORS           	-	ms6011-20	group:Keto	
#COLLECTION
CO:COLLECTION_SUMMARY            	Uninjured or SCI mice will be randomly assigned to one of four groups, LF (low
CO:COLLECTION_SUMMARY            	fat diet); HF (high fat diet); HFHS (high fat high sucrose diet); and Keto
CO:COLLECTION_SUMMARY            	(ketogenic diet). All diets will be obtained from Research Diets, NJ USA43,45.
CO:COLLECTION_SUMMARY            	Dietary fat supplementation will be initiated at 1 week after SCI. This time
CO:COLLECTION_SUMMARY            	point for intervention was chosen to provide a meaningful timeframe for clinical
CO:COLLECTION_SUMMARY            	translation. Also, a 1 week period will allow time for mice to recover prior to
CO:COLLECTION_SUMMARY            	providing access to wheel running (Aim 2). The impact of dietary fat
CO:COLLECTION_SUMMARY            	supplementation on myelin metabolism will be examined after a period of 7 weeks,
CO:COLLECTION_SUMMARY            	including determination of (i) the lipid profile of the myelin membrane using
CO:COLLECTION_SUMMARY            	LC/MS/MS; and (ii) metabolic markers of spinal cord metabolism by NMR. Results
CO:COLLECTION_SUMMARY            	will be correlated with (iii) cellular and molecular markers of spinal cord
CO:COLLECTION_SUMMARY            	pathophysiology including the appearance of OPCs, oligodendroglia and myelin,
CO:COLLECTION_SUMMARY            	axon health, astrogliosis and inflammation; and (iv) the extent of sensorimotor
CO:COLLECTION_SUMMARY            	recovery. (v) In addition, to gauge the impact of the dietary fat on systemic
CO:COLLECTION_SUMMARY            	metabolic status, Insulin and Glucose Resistance Tests will be performed at 7
CO:COLLECTION_SUMMARY            	weeks. Food intake (g/day) and body weight gain (% initial weight) will be
CO:COLLECTION_SUMMARY            	measured daily until the endpoint of each experiment. Mice will be housed
CO:COLLECTION_SUMMARY            	individually in a temperature-controlled facility with a 12:12-h light-dark
CO:COLLECTION_SUMMARY            	cycle and ad libitum access to each diet and water. A Power Analysis was
CO:COLLECTION_SUMMARY            	performed based on histological outcomes in mice with contusion compression
CO:COLLECTION_SUMMARY            	injury. To detect a difference between groups of 20%, which would very
CO:COLLECTION_SUMMARY            	meaningful, a group size of 8 will be needed to achieve a power of 0.85. An
CO:COLLECTION_SUMMARY            	additional 2 mice per group has been added to account for mortality. The Mayo
CO:COLLECTION_SUMMARY            	Clinic Institutional Animal Care and Use Committee has approved of the proposed
CO:COLLECTION_SUMMARY            	studies.
#TREATMENT
TR:TREATMENT_SUMMARY             	To test the hypothesis that optimizing dietary fat will facilitate myelin repair
TR:TREATMENT_SUMMARY             	after SCI, the diet of uninjured adult female C57BL6/J mice (12 week, 22-25g,
TR:TREATMENT_SUMMARY             	Jackson), or those with experimental contusion-compression SCI of the
TR:TREATMENT_SUMMARY             	lumbosacral spinal cord (L2-L3) (Fejota Clip 3g Force, applied for 30s)32,47
TR:TREATMENT_SUMMARY             	will be supplemented with saturated fat. The 3g Clip produces moderate SCI
TR:TREATMENT_SUMMARY             	including demyelination and clinical impairment and we recently published a
TR:TREATMENT_SUMMARY             	detailed methodology. At 1 week after injury, the 3g injured mice are expected
TR:TREATMENT_SUMMARY             	to have an average Basso Mouse Scale score (BMS)=5 on a 9 point scale such that
TR:TREATMENT_SUMMARY             	they have frequent plantar stepping with no or some coordination. This level of
TR:TREATMENT_SUMMARY             	impairment was chosen to provide a sufficient window to observe recovery, and to
TR:TREATMENT_SUMMARY             	be at a level compatible with examination of exercise training by wheel running
TR:TREATMENT_SUMMARY             	(Aim 2).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	"Myelin galactosyl sphinolipid concentrations from mouse spinal cord Lipids will
SP:SAMPLEPREP_SUMMARY            	be quantified in myelin isolated in high yield and purity by subcellular
SP:SAMPLEPREP_SUMMARY            	fractionation from the lumbosacral spinal cord. While there are no absolutely
SP:SAMPLEPREP_SUMMARY            	‘myelin-specific’ lipids, galactocerebroside is the most typical of myelin
SP:SAMPLEPREP_SUMMARY            	in the adult nervous system being directly proportional to the amount of myelin.
SP:SAMPLEPREP_SUMMARY            	Sulfatide is another galactolipid enriched in myelin. Together with cholesterol,
SP:SAMPLEPREP_SUMMARY            	these form 78% of the total amount of lipid in the myelin membrane and each will
SP:SAMPLEPREP_SUMMARY            	be quantified using LC/MS/MS. A highly sensitive assay for galactocerebroside
SP:SAMPLEPREP_SUMMARY            	was recently established by the Mayo Metabolomics Core and can be implemented
SP:SAMPLEPREP_SUMMARY            	immediately. The LC/MS/MS panel for free fatty acids, including the very long
SP:SAMPLEPREP_SUMMARY            	chain fatty acids found in myelin is also routinely performed by the Core.
SP:SAMPLEPREP_SUMMARY            	Cholesterol will be quantified using an NMR-based approach by the Mayo Dept. of
SP:SAMPLEPREP_SUMMARY            	Laboratory Medicine Clinical Core. Additionally, we have a plan in place with
SP:SAMPLEPREP_SUMMARY            	the Metabolomics Core to develop LC/MS/MS assays for sulfatide and sphingomyelin
SP:SAMPLEPREP_SUMMARY            	during the Pilot proposal. Having quantitative assays for each of these key
SP:SAMPLEPREP_SUMMARY            	myelin lipids will facilitate our goal to comprehensively profile myelin lipid
SP:SAMPLEPREP_SUMMARY            	metabolism and will form foundational assays for a future NIH grant focused on
SP:SAMPLEPREP_SUMMARY            	myelin metabolism."
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	AMT HALO HILIC 2.7, 4.6x150mm
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Quantiva QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	MALDI
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	ng/vial
MS_METABOLITE_DATA_START
Samples	ms6011-1	ms6011-2	ms6011-3	ms6011-4	ms6011-5	ms6011-6	ms6011-7	ms6011-8	ms6011-9	ms6011-10	ms6011-11	ms6011-12	ms6011-13	ms6011-14	ms6011-15	ms6011-16	ms6011-17	ms6011-18	ms6011-19	ms6011-20
Factors	group:LFD	group:LFD	group:LFD	group:LFD	group:LFD	group:HFD	group:HFD	group:HFD	group:HFD	group:HFD	group:HFHS	group:HFHS	group:HFHS	group:HFHS	group:HFHS	group:Keto	group:Keto	group:Keto	group:Keto	group:Keto
Gal-Sph	not found	not found	not found	not found	0.77	not found	0.42	0.32	0.76	1.86	not found	0.75	0.35	not found	0.80	not found	0.82	0.88	0.82	not found
Gal-C16-Cer	5	7	10	6	7	7	5	7	9	15	7	10	5	8	8	6	10	7	8	5
Gal-C18-Cer	292	364	494	287	353	342	261	357	461	706	316	497	245	424	418	311	516	363	449	251
Gal-C24:1-Cer	2379	2788	3318	2521	2747	2697	2225	2893	3277	4646	2737	3430	2305	3007	3220	2498	3545	2741	3147	2361
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Gal-Sph
Gal-C16-Cer
Gal-C18-Cer
Gal-C24:1-Cer
METABOLITES_END
#END