EPA and DHA levels in whole blood decrease more rapidly when stored at -20 °C as compared with room temperature, 4 and -75 °C.
Author(s): Metherel AH, Aristizabal Henao JJ, Stark KD
Publication: Lipids, 2013, Vol. 48, Page 1079-91
PubMed ID: 23949919 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of dietary fish oil supplementation, storage temperature and duration, treatment with different anticoagulants and the additive butylated hydroxytoluene (BHT) on fatty acid profiles in blood spots and blood stored in cryovials.
Conclusion of Paper
Fish oil supplementation increased the concentration and weight percentage eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA), percentage omega-3 highly unsaturated fatty acids (HUFA) and decreased the omega-6/omega-3 fatty acid ratio and levels of 6 of 7 polyunsaturated fatty acids (PUFA). Generally, EPA+DHA concentrations were more stable when stored for 60 days or less at room temperature, 4ºC or -75ºC as blood spots than blood in vials, but the opposite was true when storage was at -20ºC. EPA+DHA concentrations were most stable when filter paper was pre-treated with BHT and blood spots were stored at -75ºC. Declines in EPA+DHA occurred most rapidly when specimens were storedas blood spots on untreated filter paper at -20ºC., It is important to note that significant declines in EPA+DHA concentrations only occurred after at least 60 days at room temperature, regardless of whether the additive BHT was used. When blood was stored in cryovials EPA+DHA concentrations were most stable when blood was treated with heparin and BHT compared to EDTA alone, although the percentage of EPA+DHA by weight still declined rapidly. Finally, the percentage of omega-3 fatty acids by weightt was more stable than the percentage EPA+DHA.
Studies
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Study Purpose
The purpose of this study was to compare the stability of EPA+DHA concentrations in blood spots and fresh or frozen blood after storage for up to 180 days at room temperature, 4ºC, -20ºC, or -75ºC. EDTA blood obtained from a single healthy male was pipetted onto Whatman paper that was pretreated with 0 or 50 µg BHT, and then stored prior to drying. Additional venous blood was collected from the same individual and was aliquoted prior to storage.
Summary of Findings:
Percentages of arachidonic acid, EPA, omega-3 docosapentaenoic acid, DHA and omega-3 PUFA were higher in whole blood than blood spots. EPA+ DHA concentrations were more stable in blood stored as spots than in vials when storage occurred at room temperature, 4ºC or -75ºC for 60 days or less (p<0.05, all at 60 days). When specimens were stored at -20ºC, EPA+ DHA concentrations were more stable when blood was stored in vials than as spots regardless of storage duration. Further, when specimens were stored for 180 days concentrations were higher in blood stored in vials than as spots at room temperature, -20°C, or -80°C (p<0.05, all).
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid GC- flame ionization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Refrigeration
Storage Storage conditions On Whatman paper
In vials
Storage Storage duration 0 days
1 day
3 days
7 days
14 days
30 days
60 days
90 days
120 days
150 days
180 days
Storage Storage temperature Room temperature
4ºC
-20ºC
-75ºC
-
Study Purpose
The purpose of this study was to determine the effects of omega-3 dietary supplements, filter paper treatment with BHT, and storage temperature and duration on EPA+DHA concentrations in blood spots. EDTA blood was obtained from a single healthy male before (low omega-3) and after (high omega-3) fish oil supplementation for 2 months. Blood was pipetted onto Whatman paper that was pretreated with 0 or 50 µg BHT, and then stored prior to drying. Effects of storage were verified using blood from 10 healthy individuals pipetted onto Whatman paper without BHT.
Summary of Findings:
Fish oil supplementation led to a 297% increase in the percentage EPA+DHA, a 215% increase in the percentage of omega-3 HUFA, and a 61% decrease in the omega-6/omega-3 ratio in comparison to specimens collected prior to dietary supplementation. Further, levels of 6 of the 7 omega-6 PUFA assayed (including arachidonic and linoleic acid) decreased after dietary supplementation compared to controls obtained beforehand. Blood spotted onto untreated filter paper prior to dietary supplementation exhibited declines in EPA+DHA concentrations after only 1 day at -20ºC, after 60 days at room temperature, 7 days at 4ºC, and 180 days at -75ºC. Similarly, blood spotted onto untreated filter paper after dietary supplementation with high omega-3 levels, exhibited declines in EPA+DHA concentrations after 3 days at -20ºC, 120 days at room temperature, 60 days at 4ºC, and 7 days at -75ºC, but the decline that occurred at -75ºC was only noted at 7 days not when stored for 14 days or more. When blood was spotted onto filter paper pre-treated with BHT, EPA+DHA concentrations remained stable for longer, as declines were reported after 60 days at room temperature or 180 days at -75 ºC, regardless of dietary supplementation, and after 60 days at 4ºC or -20ºC in blood collected prior to dietary supplementation, and after 120 days at 4ºC or 7 days at -20ºC in blood collected after dietary supplementation. Importantly, blood spots collected from all 10 individuals displayed similar EPA+DHA stability in response to different storage durations and temperatures
Biospecimens
Preservative Types
- Frozen
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid GC- flame ionization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Patient diet After 2 months of fish oil supplementation
No fish oil supplementation
Storage Storage duration 0 days
1 day
3 days
7 days
14 days
30 days
60 days
90 days
120 days
180 days
Biospecimen Preservation Fixative additive/buffer Butylated hydroxytoluene
None
Storage Storage temperature Room temperature
4ºC
-20ºC
-75ºC
Preaquisition Biomarker level Low omega-3
High omega-3
Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Refrigeration
-
Study Purpose
The purpose of this study was to compare EPA+DHA concentrations and weight in whole blood treated with different anticoagulants (heparin or EDTA). Venous blood from a single healthy individual was collected into tubes that contained one of three anticoagulant combinations (EDTA, heparin alone, or heparin and BHT). Specimens were aliquoted prior to storage at one of four different temperatures.
Summary of Findings:
A significant effect of anticoagulant and/or BHT treatment was found for all storage temperatures examined. Generally, blood treated with heparin exhibited less severe declines in EPA+DHA concentrations than EDTA. While differences in EPA+DHA concentrations between anticoagulants were largest when specimens were stored at -20ºC, significant declines in concentration and weight were noted at all temperatures. When blood was treated with both BHT and heparin, EPA+DHA stability at room temperature was extended from 14 days (heparin alone) to 180 days; however, significant declines in the percentage of EPA+DHA by weight were observed after 3 or more days of storage regardless of anticoagulant and/or BHT. When BHT was added to heparin treated blood, declines in EPA+DHT concentrations occurred faster during frozen storage than when BHT was omitted. While EPA+DHT concentrations were lower after storage of heparin or EDTA blood without BHT for 60 days or more at -20ºC or -75ºC, this effect was not observed in heparin blood with BHT even after 180 days at -20ºC or -75ºC. However, addition of BHT did not increase the stability of the EPA+DHT weight in heparin blood at -20ºC and resulted in a decrease in EPA+DHT weight after storage at -75ºC for 180 days that was not found when BHT was omitted.
Biospecimens
Preservative Types
- Frozen
- Other Preservative
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid GC- flame ionization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Refrigeration
Biospecimen Preservation Fixative additive/buffer None
Butylated hydroxytoluene
Biospecimen Acquisition Anticoagulant EDTA
Heparin
Storage Storage duration 0 days
1 day
3 days
7 days
14 days
30 days
60 days
90 days
120 days
150 days
180 days
Storage Storage temperature Room temperature
4ºC
-20ºC
-75ºC
-
Study Purpose
The purpose of this study was to determine the effects of the additive BHT on the percentage omega-3 fatty acids by weight in relation to total HUFA after storage at different temperatures and durations. EDTA blood obtained from a single healthy male was pipetted onto Whatman paper that was pretreated with 0 or 50 µg BHT, and then stored prior to drying. Additional venous blood from the same individual was collected into the three anticoagulant combinations (EDTA, heparin, or heparin and BHT) and aliquoted prior to storage at room temperature, 4ºC, -20ºC, or -75ºC for different durations.
Summary of Findings:
The percentage of omega-3 fatty acids by weight in relation to total HUFA was stable for at least 7 days, regardless of storage temperature, addition of BHT and anticoagulant, and was generally more stable in cryovials than in blood spots. The percentage of omega 3 fatty acids by weight was most stable in blood spots when stored at -75ºC and least stable when stored at -20ºC, although stability at -20ºC was improved when filter paper was pre-treated with the additive BHT. When blood was stored in cryovials the percentage of omega-3 fatty acids by weight was more stable when anticoagulated with heparin as opposed to EDTA, and tended to be more stable when stored at -20ºC or -75ºC compared to 4ºC or room temperature.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Lipid GC- flame ionization Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation None (fresh)
Frozen
Refrigeration
Biospecimen Preservation Fixative additive/buffer Butylated hydroxytoluene
None
Storage Storage conditions On Whatman paper
In vials
Storage Storage duration 0 days
1 day
3 days
7 days
14 days
30 days
60 days
90 days
120 days
150 days
180 days
Storage Storage temperature Room temperature
4ºC
-20ºC
-75ºC