Blood sphingolipidomics in healthy humans: impact of sample collection methodology.
Author(s): Hammad SM, Pierce JS, Soodavar F, Smith KJ, Al Gadban MM, Rembiesa B, Klein RL, Hannun YA, Bielawski J, Bielawska A
Publication: J Lipid Res, 2010, Vol. 51, Page 3074-87
PubMed ID: 20660127 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of fasting prior to specimen collection, freeze-thaw cycling, gender, and anticoagulant on sphingolipid levels in serum and plasma.
Conclusion of Paper
While higher levels of certain sphingolipids were measured in specimens from females than those from males, the influence of fasting conditions on sphingolipid levels was generally similar for both genders. Fasting resulted in significantly higher levels of the sphingomyelin (SM) species C16-SM, long-chain LacCers, and C26-Cer1P, but lower levels of short-chain LacCers, and short- and medium-chain Cers. The authors state that generally, sphingolipids in serum and plasma specimens showed the same distributions and responses to fasting conditions, regardless of anticoagulant. However, C16-HexCer was higher in nonfasting serum and EDTA-plasma specimens compared to fasting specimens, but lower in nonfasting heparinated plasma compared to fasting heparinated plasma. Levels of S1P and dhS1P were significantly higher in serum than in plasma, but there were no significant differences in S1P or dhS1P levels between citrated, heparinated, or EDTA-plasma. After 3 or 4 freeze-thaw cycles, recovery of some sphingolipids from serum or EDTA-plasma was inconsistent.
Studies
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Study Purpose
The purpose of this study was to determine the effects of fasting prior to specimen collection, gender, and anticoagulant on sphingolipid levels in serum and plasma. All plasma and serum specimens were frozen at -80 degrees C until lipid extraction and analysis.
Summary of Findings:
Higher levels of certain sphingolipids were measured in specimens from females than those from males, however, the influence of fasting conditions on sphingolipid levels was generally similar for both genders. The most abundant SM species (C16-SM) was significantly higher in serum from fasting individuals than specimens taken from nonfasting individuals, while other SM species were unaffected by fasting. Fasting resulted in significantly higher levels of long-chain LacCers and lower levels of short-chain LacCers. The authors state there were no significant differences in the distributions of SM or LacCer species and responses to fasting conditions between serum and plasma, regardless of anticoagulant. The most abundant HexCer species (C24-HexCer) was not significantly different between fasting or nonfasting specimens for serum, EDTA-plasma, or heparinated plasma. However, C16-HexCer was higher in nonfasting serum and EDTA-plasma specimens compared to fasting specimens but lower in nonfasting heparinated plasma compared to fasting heparinated plasma. No significant differences were seen in levels of Cer species between serum and plasma, regardless of anticoagulant. Levels of S1P and dhS1P were significantly higher in serum than in plasma, but there were no significant differences in S1P or dhS1P levels between citrated, heparinated, or EDTA-plasma. Short- and medium-chain Cers decreased under fasting conditions, while C26-Cer1P levels increased in response to fasting, and the authors state that these changes were seen for all plasma preparations and serum. Overall, EDTA-plasma gave the most similar results, in terms of sphingolipid levels, compared to serum (no anticoagulant) and was less variable than heparinated or citrated plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid HPLC-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Time of biospecimen collection After 10 h fast
2 h after standard meal
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Preaquisition Patient gender Female
Male
Biospecimen Acquisition Anticoagulant EDTA
None
Sodium citrate
Sodium heparin
-
Study Purpose
The purpose of this study was to determine the effects of freeze-thaw cycling on sphingolipid levels in serum and EDTA-plasma from fasting males. All plasma and serum specimens were frozen at -80 degrees C. The second, third, and forth thaw cycles were performed on three consecutive days, the first two thaws occurring at room temperature for 3 h, and the last occurring overnight at 4 degrees C.
Summary of Findings:
There were no statistically significant differences in sphingolipid levels between specimens that underwent 1 and 2 freeze-thaw cycles. However, after 3 or 4 freeze-thaw cycles, recovery of some sphingolipids was inconsistent. The authors state that some of the variability was due to lower internal standard recovery. EDTA-plasma that underwent 4 total freeze-thaw cycles showed more variability in the measurement of individual sphingolipids than serum that underwent the same number of freeze-thaw cycles.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid HPLC-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Freeze/thaw cycling 1 cycle
2 cycles
3 cycles
4 cycles
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum