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Impact of post-surgical freezing delay on brain tumor metabolomics.

Author(s): Mock A, Rapp C, Warta R, Abdollahi A, Jäger D, Sakowitz O, Brors B, von Deimling A, Jungk C, Unterberg A, Herold-Mende C

Publication: Metabolomics, 2019, Vol. 15, Page 78

PubMed ID: 31087206 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper assessed the effects of cold ischemia time on the metabolome (and levels individual metabolites and metabolic pathways) in glioblastoma specimens using ultrahigh performance liquid chromatography/ tandem mass spectrometry (UHPLC-MS/MS) and gas chromatography/mass spectrometry (GC-MS). A total of 34 tumor specimens were collected from three patients with glioblastomas (GBMs).  Each tumor piece was divided into 10-12 specimens by a pathologist and subjected to a cold ischemia time of 0 min (preserved within 30 s of resection), 5, 10, 30, 60, and 180 min, then snap-frozen in 2-methyl-butane that was pre-cooled with liquid nitrogen, transferred to a cryotube and stored at -80°C.  Tumor cell content and necrosis were confirmed to be >80% and <20%, respectively, by hematoxylin and eosin staining OCT-embedded tissue specimens. Frozen tissue specimens were sent to a commercial lab for UHPLC-MS/MS and GC-MS analysis, and the resultant data was processed and normalized with the MetaboDiff R package. A linear mixed effects model was employed to identify significant differences in metabolic measurements due to a delay to preservation.

   Summary of Findings:

   The assessment of tissue integrity by microscopic analysis of H&E-stained slides resulted in the exclusion of two specimens (both specimens had a cold ischemia time of 30 min) from subsequent analysis due to low tumor content and a percentage of necrotic cells that exceeded the threshold (<20% necrosis).  Normalized metabolic measurements of individual samples clustered by patient, not cold ischemia time, after both principal component analysis and hierarchical clustering, indicating that the variance between patients was greater than the variance introduced by the cold ischemia times investigated.

   
   During the cold ischemia timecourse, 10% (59/597) of the named metabolites detected in the glioblastoma specimens displayed a significant change in abundance (P<0.05), leading the authors to conclude that changes in metabolites were not system-wide. The authors conclude that the changes are indicative of active metabolism based on an increase in 31 metabolites (including peptides and lipids) and a decrease in 28 metabolites (including carbohydrates and energy metabolites).

   
   Fold-changes of individual metabolites in glioblastoma specimens that occurred at each timepoint over the cold ischemia time course were provided in the article; for example, "glucose decreased by 10% for every 10 min delay".  Changes were also summarized for each metabolite class, with the largest differences (an approximate doubling of the relative concentration after 150 min) affecting "peptides, energy metabolites, and carbohydrates"; amino acids were relatively stable during the cold ischemia timecourse.  
    

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Mixed type

   Platform:

   Analyte	Technology Platform
   Small molecule	GC-MS
   Small molecule	LC-MS or LC-MS/MS
   Lipid	LC-MS or LC-MS/MS
   Peptide	LC-MS or LC-MS/MS
   Carbohydrate	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 min (≤30 s) 5 min 10 min 30 min 60 min 180 min

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