The Impact of Freezing Temperature and Pretreatment Protocol on Solid Phase HR-MAS-MRS Metabolomes of Colorectal Cancer Tissue

Background: To explore the best pretreatment method of colorectal cancer tissue samples for metabolomics research based on solid-phase nuclear magnetic resonance. Method: Taking mucosal tissues of colorectal cancer and divide it into 5 groups of 0.2cm*0.2cm*0.2cm. Pretreatment was performed as follows: I. Liquid nitrogen storage; II. Transfer to the -80℃ refrigerator after storing in liquid nitrogen for 10 minutes; III. Transfer to the -80℃ refrigerator after storing in liquid nitrogen for 20 minutes; IV. Transfer to the -80℃ refrigerator after storing in liquid nitrogen for 30 minutes; V. -80℃ refrigerator storage. The interval between tumor sample separation to pretreatment is less than 30 minutes. The tissue sample testing process is carried out on Bruker AVII-600 Spectrometer equipped with a high-resolution probe having a 1H/13C magical angle rotation. The tissue samples were put into the NMR which run at a speed of 5000Hz for 10 minutes. NMR signals were collected and analyzed by Fourier transform, partial least squares discrimination analysis (PLS-DA). Corresponding metabolites and metabolic pathways were found in Human Metabolome Database (HMDB) according to the ppms with variable importance of projection (VIP) ＞1. Results: The content of pelargonic acid, stearic acid, D-Ribose, heptadecanoic acid, pyruvic acid, succinate, sarcosine, glycine, creatine, and L-lactate in liquid nitrogen storage group were significantly lower than the other groups (P<0.05), the content of glycerophosphocholine in liquid nitrogen storage group was lower than the other groups (P=0.055). Pyruvic, succinate and L-lactate are participating in glucose metabolism. Glycerophosphocholine, sarcosine, glycine and creatine are participating in choline phospholipid metabolism. This indicated that the glucose and choline phospholipid metabolism levels of the liquid nitrogen group were significantly lower than those of the other 4 groups. Conclusion: Liquid nitrogen storage can slow down the glucose and choline phospholipid metabolism process of colorectal cancer tissue samples in vitro; liquid nitrogen can preserve tissue sample’s metabolic state in the body. It is therefore the better way to store tissue sample than the other methods. clinical trial registry website: http://www.chictr.org.cn/index.aspx. Trial number: ChiCTR1900024640