Effects of sample handling and storage on quantitative lipid analysis in human serum

Abstract

There is sparse information about specific storage and handling protocols that minimize analytical error and variability in samples evaluated by targeted metabolomics. Variance components that affect quantitative lipid analysis in a set of human serum samples were determined. The effects of freeze-thaw, extraction state, storage temperature, and freeze-thaw prior to density-based lipoprotein fractionation were quantified. The quantification of high abundance metabolites, representing the biologically relevant lipid species in humans, was highly repeatable (with coefficients of variation as low as 0.01 and 0.02) and largely unaffected by 1-3 freeze-thaw cycles (with 0-8% of metabolites affected in each lipid class). Extraction state had effects on total lipid class amounts, including decreased diacylglycerol and increased phosphatidylethanolamine in thawed compared with frozen samples. The effects of storage temperature over 1 week were minimal, with 0-4% of metabolites affected by storage at 4°C, -20°C, or -80°C in most lipid classes, and 19% of metabolites in diacylglycerol affected by storage at -20°C. Freezing prior to lipoprotein fractionation by density ultracentrifugation decreased HDL free cholesterol by 37% and VLDL free fatty acid by 36%, and increased LDL cholesterol ester by 35% compared with fresh samples. These findings suggest that density-based fractionation should preferably be undertaken in fresh serum samples because up to 37% variability in HDL and LDL cholesterol could result from a single freeze-thaw cycle. Conversely, quantitative lipid analysis within unfractionated serum is minimally affected even with repeated freeze-thaw cycles.

Preanalytical Aspects and Sample Quality Assessment in Metabolomics Studies of Human Blood

“…Selected preanalytical aspects of targeted metabolomics investigations of human blood and urine by GC-MS and LC-MS (11)(12)(13)(14)(15)(16)(17), as well as the effects of delayed storage on human cerebrospinal fluid metabolomes (18 ), have been reported. Recently, protocols for metabolic profiling of serum and plasma have been published (3,7,19 ).…”

No Apparent Impact of Meal Glycemic Index on Postprandial Lipid Response in Lean and Abdominally Obese Women

“…Freezing of the plasma samples could impact the lipid results we obtained [48]. Plasma triglyceride levels are also subject to greater physiological variation than cholesterol levels [49].…”

Serum fatty acid profiling within distinct lipid fractions provides a more robust indicator of insulin resistance in humans than total triglyceride and fatty acid profiles

“…many variables may yet be unknown or "uncontrollable") it remains important to recognize and consider these variables when establishing an experimental design. Failure to do so can influence results and potentially lead to an inappropriate interpretation of the data [313,364]. Therefore, the following sections will highlight recent knowledge that is pertinent for nutritional metabolomics (summarized in Table 1).…”

“…Investigating the influence of freeze--thaw cycling and temperature storage on lipid profiles, Zivkovic et al found that samples exposed to multiple freeze--thaw cycles had little impact on the lipid profile (≤ 4% variability); however, diacylglycerol concentrations in particular decreased by 19% after one week in samples stored at --20°C compared to --80°C [364]. Therefore, to maintain metabolite integrity and minimize degradation, samples…”