Effect of Repeated Freeze-Thaw Cycles on NMR-Measured Lipoproteins and Metabolites in Biofluids.

Metabolic profiling of biofluids by nuclear magnetic resonance (NMR) spectroscopy serves as an important tool in disease characterization, and its accuracy largely depends on the quality of samples. We aimed to explore possible effects of repeated freeze-thaw cycles (FTCs) on concentrations of lipoprotein parameters in serum and metabolite concentrations in serum and urine samples. After one to five FTCs, serum and urine samples (n= 20) were analyzed by NMR spectroscopy, and 112 lipoprotein parameters, 20 serum metabolites, and 35 urine metabolites were quantified by a commercial analytical platform. Principal component analysis showed no systematic changes related to FTCs, and samples from the same donor were closely clustered, showing a higher between-subject variation than within-subject variation. The coefficients of variation were small (medians of 4.3%, 11.0%, and 4.9%  for lipoprotein parameters and serum and urine metabolites, respectively). Minor, but significant accumulated freeze-thaw effects were observed for 32 lipoprotein parameters and one serum metabolite (acetic acid) when comparing FTC1 to further FTCs. Remaining lipoprotein and metabolite concentrations showed no significant change. In conclusion, five FTCs did not significantly alter the concentrations of urine metabolites and introduced only minor changes to serum lipoprotein parameters and metabolites evaluated by the NMR-based platform.