Alterations in Human Liver Metabolome during Prolonged Cryostorage.
Author(s): Abuja PM, Ehrhart F, Schoen U, Schmidt T, Stracke F, Dallmann G, Friedrich T, Zimmermann H, Zatloukal K
Publication: J Proteome Res, 2015, Vol. 14, Page 2758-68
PubMed ID: 26036795 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine if temperature fluctuations during cryostorage, such as those that occur when samples are retrieved, and the number of times these events occur adversely affect the liver metabolome. Potential impacts of storage temperature on the liver metabolome were also explored.
Conclusion of Paper
Inter-specimen variability in the metabolome was much greater than intra-specimen variability, regardless of the number of retrieval cycles or storage temperature (liquid nitrogen, nitrogen vapor or -80ºC freezer). However, 41 metabolites displayed significant effects following 1000 retrieval cycles, although the magnitude of change remained within 15% for all specimens, and within 5% for most. When specimens were cycled 10 times, but maintained at the retrieval temperature until calculated to reach a specified internal temperature, significant changes in many lipid levels occurred when core temperatures reached -60ºC, while most non-lipids were stable until core temperatures exceeded -20ºC.
Studies
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Study Purpose
This study investigated whether the liver metabolome is affected by the temperature of frozen storage; and the temperature, duration and number of cycles specimens are exposed to during sample retrieval from freezers of nitrogen dewars, as well as the core temperature of the specimen itself during such retrievals. Non-tumorous specimens from 5 patients with liver metastasis of non-hepatic primary tumors were snap-frozen in isopentane and transferred into liquid nitrogen. All transport was done in a dry shipper. Specimens were subjected to 300, 1000, 3000 or 10,000 transfers between storage (liquid nitrogen, nitrogen vapor or -80ºC freezer) and retrieval temperatures (room temperature, or -80ºC freezer) using a cable pull-system or a robotic cycler. The authors state that the number of cycles (300, 1000, 3000 or 10,000) corresponds to 3 months, 1 year, 3 years and 10 years of typical storage, respectively. Specimens were at the storage temperature for up to 8 min and the retrieval temperature for up to 90 s. Additionally, potential effects associated with the duration of the retrieval event were also investigated. Specimens were maintained at a retrieval temperature of 20°C for unspecified durations which reflected an internal specimen temperature of -60ºC, -40ºC, -20ºC, 0ºC, or 10ºC using pig liver. After cycling all specimens were stored in liquid nitrogen vapor until homogenization and analysis by LC-MS.
Summary of Findings:
Inter-specimen variability in metabolite levels was much greater than intra-specimen variability, regardless of the number of retrieval cycles or storage in liquid nitrogen, nitrogen vapor or -80ºC freezer at. When metabolite levels for each specimen were normalized to a control (stored in liquid nitrogen vapor for the duration of the experiment), 41 metabolites displayed a significant effect after 1000 cycles (p<0.01), although changes were equal or less than 15% for all specimens and equal or less than 5% for most. In general, 1000 retrieval cycles led to increases in amino acids and biogenic amines, and decreases in sulfuroamino acids. Effects of cycling on acylcarnitines, glycerophospholipids, and prostaglandins were small and may be dependent on storage temperature, as smaller declines were generally noted at lower temperatures (liquid nitrogen or nitrogen vapor vs -80°C. Although the authors state that changes in proline remained less than15% even after 10,000 cycles, they recommended a maximum of 1000 cycles. Changes in temperature during retrieval occurred predominantly at the wall of the specimen vial, not at the air-interface. When specimens were kept at the retrieval temperature for longer than 90 s, significant changes in the levels of many lipids were noted when core temperatures were allowed to reach -60ºC, but most non-lipids were stable unless core temperatures exceeded -20ºC, with the exceptions of several acylcarnitines, sarcosine, leukotriene B4, homocysteine, and glutathione disulfide.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Normal Adjacent
Platform:
Analyte Technology Platform Small molecule LC-MS or LC-MS/MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature -196ºC (liquid nitrogen)
-150ºC (liquid nitrogen vapor)
-80ºC (freezer)
Storage Freeze/thaw cycling 300 cycles
1000 cycles
3000 cycles
10000 cycles
Storage Thaw duration <90 s
Until calculated to reach -60ºC
Until calculated to reach -40ºC
Until calculated to reach -20ºC
Until calculated to reach 0ºC
Until calculated to reach 10ºC
Storage Thaw temperature/condition -80ºC
Room temperature