Pre-Analytical Factors that Affect Metabolite Stability in Human Urine, Plasma, and Serum: A Review

Metabolomics provides a comprehensive assessment of numerous small molecules in biological samples. As it integrates the effects of exogenous exposures, endogenous metabolism, and genetic variation, metabolomics is well-suited for studies examining metabolic profiles associated with a variety of chronic diseases. In this review, we summarize the studies that have characterized the effects of various pre-analytical factors on both targeted and untargeted metabolomic studies involving human plasma, serum, and urine and were published through 14 January 2019. A standardized protocol was used for extracting data from full-text articles identified by searching PubMed and EMBASE. For plasma and serum samples, metabolomic profiles were affected by fasting status, hemolysis, collection time, processing delays, particularly at room temperature, and repeated freeze/thaw cycles. For urine samples, collection time and fasting, centrifugation conditions, filtration and the use of additives, normalization procedures and multiple freeze/thaw cycles were found to alter metabolomic findings. Consideration of the effects of pre-analytical factors is a particularly important issue for epidemiological studies where samples are often collected in nonclinical settings and various locations and are subjected to time and temperature delays prior being to processed and frozen for storage.

Tube additives [6] Methionine sulfoxide higher in serum from a gel-barrier tube than for tube with clot activator.
[7] Variability greater for bile acids and carbohydrates in heparin plasma than in EDTA plasma. Details can be found in Townsend et al., Table 3. [3] Higher in citrate plasma than EDTA plasma: glutamic acid; higher in EDTA plasma than citrate plasma: sarcosine. Details can be found in Paglia et al., Table 1 and Fasting status [8] Fasting status affected taurine, acylcarnitines, PCs and sphingolipids. Details can be found in Carayol et al., Supplementary Table S1. [7] Fasting status affected bile acids, purines, pyrimidines, and vitamins. [9] Fasting status affected bile acids and vitamins.
Collection time of day and season [12] Time of day of blood collection affected acylcarnitines, lysophospholipids, bilirubin, corticosteroids, and amino acids. Details can be found in Ang et al., Table 1. [9] Time of day of blood collection affected bile acids and vitamins. [9] Season of blood collection affected bile acids, purines, pyrimidines and organic acids.
Pre-centrifugation time delay and temperature [13] Delayed processing for up to 24 h at either 4⁰C or 25⁰C affected glucose, pyruvate, and lactate. [14] Delayed for up to 8 h at 4⁰C for up to 8 h affected lactate, pyruvate, and glucose.   Delays of up to 24 h at either 4⁰C or room temperature affected lactate and glucose. [17] Delays of up to 6 h at room temperature affected aspartate, glutamate, and methionine. [18] Delays of up to 20 h at room temperature affected metabolites involved in glycolysis/gluconeogenesis/pyruvate metabolism, phospholipid and purine metabolism, the TCA cycle, fatty acids, amino acids and other metabolites. Details can be found in Jain et al., Figures 1-3 and Table 2.
Delays of up to 6 h at 4⁰C or 22⁰C affected lactate, glucose, fatty acids, choline and acetone. Details can be found in Jobard et al., Table S1. [10] Delays of up to 2 h at room temperature or up to 6 h on wet ice affected carbohydrates and lipids. Details can be found in Kamalage et al. 2014, Table 3. [20] Delays of up to 6 h at room temperature affected amino acids, carbohydrates, energy metabolism metabolites, nucleobases, vitamins, cofactors, and other metabolites. Details can be found in Kamlage et al. 2018 Table 1, Figures 2, 3, 4, and 6, and  Supplemental Tables S1 and S2. [21] Delays up to 24 h at either 4⁰C or 23⁰C affected amino acids, carboxylic acids, fatty acids, fatty alcohols, monosaccharides, sugar acids, inositol, palmitic acid, lactic acid, uric acid, α-ketoglutaric acid, creatinine, adenosine-5-monophosphate, cholesterol, taurine, and urea. Details can be found in Malm et al., Figure 2.
Delays up 48 h on ice affected B vitamins, amino acids, kynurenines, other metabolites. Details can be found in Midttun et al., Figure 1.
Delays of up to 30 minutes at room temperature or up to 8 h at cold temperatures affected kynurenine, pyruvate, and organic acids. Details can be found in Nishiumi et al., Figures 3 and S1. [23] Delays of up to 23 h at either 4⁰C or room temperature affected lactic acid and ascorbic acid. [7] Delays of up to 48 h on ice packs affected carbohydrates, purines, and pyrimidines. [24] Delays of up to 48 h at 4⁰C affected nucleotides. Details can be found in Wang et al., Figure 1. [25] Delays of up to 8 h at 4⁰C affected oleoylethanolamine, anandamide, palmitoylethanolamine, and docosahexaenoylethanolamine. [11] Delays of up to 24 h at room temperature affected hypoxanthine, sphingosine-1phosphate, and linolenyl carnitine. Centrifugation conditions [26] Centrifugation speed affected glycerophosphocholines and sphingomyelins.
Post-processing time delay and temperature [27] Post-processing delays of up to 36 h at room temperature affected acylcarnitines, amino acids, lyosPCs and PCs. Details can be found in Anton et al., Table 1. [28] Post-processing delays of up to 36 h at 4⁰C affected trimethylamine-N-oxide (TMAO). [13] Post-processing delays of up to 24 h at room temperature affected albumin, triglycerides, LDL/VLDL, proline, citrate and histidine. [17] Post-processing delays of up to 60 minutes at room temperature affected tyrosine, serine, phenylalanine, aspartic acid, isoleucine, glutamate, methionine, and cysteine.
Details can be found in Hirayama et al., Supplementary Table S3. [10] Post-processing delays of up to 16 h at 4⁰C, 12⁰C, or room temperature affected signaling metabolites and amino acids. Details can be found in Kamlage et al. 2014, Table 2. [20] Post-processing delays of up to 24 h at room temperature affected ribose, aspartate, and phenylalanine. [29] Post-processing delays of up 24 h at room temperature or 1 week at 4⁰C affected eicosapentaenoate, 2-stearoylglycerophosphocholine, glycerophosphocholine, other lipids, cysteine, Ƴ-glutamyl amino acids, 5-ocoproline, N 6 -methyladenosine and allantoin. Details can be found in Moriya et al., Supplemental Figure 2.
Storage time [30] Storage for up to 5 years at -80⁰C affected amino acids, hexoses, acylcarnitines and lipids. Details can be found in Haid et al., Figures S1-2. [31] Storage for up to 5 years at -80⁰C affected lysoPCs, acylcarnitines, hypoxanthine, and serotonin. Details can be found in Yang et al., Figure 3.
Freeze/thaw cycles [27] Up to four freeze/thaw cycles affected glycine, methionine, phenylalanine, tryptophan and tyrosine. Details can be found in Anton et al., Supplemental Tables 1 and 2. [6] Up to two freeze/thaw cycles affected methionine sulfoxide, amino acids, PCs, and acetylornithine. Details can be found in Breier et al., Table S7. [16] Up to five freeze/thaw cycles affected choline, glycerol, methanol, and ethanol. Details can be found in Fliniaux et al., Tables 2 and 3B. [17] Up to 10 freeze/thaw cycles affected amino acids. Details can be found in Hirayama et al., Supplemental Figure 6. [32] Up to five freeze/thaw cycles affected lipids, alanine, glucose, acetone, and pyruvate. [25] Up to three freeze/thaw cycles affected arachidonic acid. [11] Up to four freeze/thaw cycles affected carnitine.

Urine Samples
Centrifugation conditions [13] Glutamate increases in samples not centrifuged prior to storage and/or analysis. [33] Centrifugation affected changes in levels observed after four weeks storage at room temperature of acetate, citrate, formate, succinate, trimethylamine, uracil, urea, creatine, creatinine, and phenylacetylglycine. Details can be found in Saude and Sykes, Tables 2 and 3.
Filtration and additives [33] Filtration affected changes in levels observed after four weeks storage at room temperature of acetate, benzoate, formate, glycine, Hippurate, lactate, succinate, trimethylamine, uracile, and urea. Details can be found in Saude and Sykes, Tables 2  and 3. [33] Addition of sodium azide affected changes in levels observed after four weeks storage at room temperature of acetate, citrate, formate, glycine, hippurate, lactate, succinate, trimethylamine, uracil, and urea. Details can be found in Saude and Sykes, Table 2.
Details can be found in Roux et al., Table 2.
Storage time and temperature [33] Storage for four weeks at room temperature affected acetate, benzoate, citrate, creatinine, formate, glycine, hippurate, lactate, malonate, succinate, trimethylamine, and urea. Details can be found in Saude and Sykes, Table 4. [37] Storage for seven days at 4⁰C affected acetate.