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Eicosanoid Sample Collection, Preparation, and Storage Advice

Article from 2021-01-20

Because of the shift in localization of lipid mediators from their tissues/cells of origin to the circulation and finally for excretion in urine, careful consideration of whole-body metabolism must be made when choosing specific sample types used for measurement. The complexity of the sample may also dictate whether or not purification is needed prior to analysis.

Prostaglandin metabolism from source to excretion.

Cell culture supernatants

Often contain high levels of bioactive lipids
Metabolism of bioactive lipids is limited, so measurement of the parent compound is appropriate
Generally do not require purification

Cell lysates

May contain higher levels of bioactive lipids than found in vivo
Metabolism of bioactive lipids is generally limited, so measurement of the parent compound is often appropriate

Plasma/Serum

Contains low levels of most bioactive lipids, the exception being those that are synthesized during the clotting process in serum preparation
Measurement of bioactive lipids in serum can provide an index of synthetic capacity of some bioactive lipids formed during the clotting process, particularly platelet thromboxane synthesis, rather than a measurement of systemically circulating compounds
Bioactive lipids are often rapidly metabolized in vivo; to gauge circulating levels of bioactive lipids it is more appropriate to measure downstream metabolites rather than the parent compound
Due to the complexity of these sample matrices and the relatively low level of bioactive lipids present, purification is advised

Tissue

Levels of bioactive lipids may be higher than found in plasma
It is often appropriate to measure the parent compound, but will depend on the tissue being tested
Due to the complexity of this sample matrix, purification is advised

Urine

Contains much higher levels of bioactive lipids than are found in plasma
The kidney is capable of producing a wide range of bioactive lipids; therefore, urine contains a mixture of bioactive lipids of both systemic and renal origin
Measurement of systemically produced bioactive lipids is best achieved by measurement of downstream metabolites to minimize the contribution of those of renal origin
Due to the relatively high levels of bioactive lipids in urine, purification is generally not necessary

Examples of eicosanoids that are appropriate to measure in different sample types:

Thromboxane A₂ (TXA₂)

Sample Type	Analyte	Notes
Cell culture supernatants and lysates	TXB₂	TXA₂ is extremely unstable and is converted non-enzymatically to TXB₂. Note: the presence of TXB₂ in FBS can be significant and should be accounted for in the final analysis; alternatively use serum-free culture conditions.
Tissue	TXB₂	There is little conversion of TXB₂ to downstream metabolites in most tissue types.
Plasma/Serum	11-dehydro TXB₂	Platelets are activated during blood collection, producing high levels of TXB₂, which will mask the signal from circulating TXB₂. Since TXB₂ will not be metabolized to 11-dehydro TXB₂ ex vivo, the levels of this metabolite should better reflect systemic levels of thromboxane.
Urine	11-dehydro TXB₂	Circulating TXB2 is rapidly metabolized in both liver and kidney.

Prostaglandin D₂ (PGD₂)

Sample Type	Analyte	Notes
Cell culture supernatants and lysates	PGD₂	Metabolism is limited in vitro, so measurement of the parent compound is appropriate.
Plasma	11β-PGF_2α	PGD₂ is rapidly metabolized to 11β-PGF_2α in vivo. It also adducts to proteins through Michael addition following dehydration to PGJ₂, so attempts to measure PGD₂ itself in plasma or urine will be inaccurate. 11β-PGF_2α is a relatively stable PGD₂ metabolite that can be measured in plasma.
Urine	tetranor-PGDM	While 11β-PGF_2α may provide useful information about urinary PGD₂ levels in some species, in other species, such as rodents, it is found at very low levels and is, therefore, not a reliable index of systemic PGD₂ formation. tetranor- PGDM is a further downstream metabolite of PGD₂, which provides a more accurate index of systemic PGD₂ levels.

Prostaglandin E₂ (PGE₂)

Sample Type	Analyte	Notes
Cell culture supernatants and lysates	PGE₂	Metabolism is limited in vitro, so measurement of the parent compound is appropriate.
Plasma	PGE Metabolite (PGEM)	PGE₂ is rapidly metabolized in vivo. PGEM provides a good tool for measurement of these downstream metabolites.
Urine	PGE Metabolite (PGEM)	The kidney is capable of PGE₂ production, therefore urinary PGE₂ is a mixture of eicosanoid produced systemically and that of renal origin. PGEM found in urine will represent only PGE₂ that is produced systemically.

Preventing Artifacts

Several different potential sources of artifacts could result in inaccurate quantification of bioactive lipids. Here are some solutions to avoid confounding variables.

Time Frame	Cause	Problem	Solution(s)
During experiment	Arachidonic acid added to cultured cells	Arachidonic acid cross-reacts at a very low level with many eicosanoid ELISAs. Although the cross-reactivity is low, the total amount of arachidonic acid added to the cells may be quite high, resulting in artificially elevated measurement of eicosanoid in the sample.	Limit the amount of exogenous arachidonic acid added to cultured cells. Most of Cayman's ELISA kit booklets for our eicosanoid assays provide cross-reactivity information for arachidonic acid. Using this information, the arachidonic acid interference can easily be diluted out.
During sample collection	Activation of platelets and endothelial cells during venipuncture	Exogenous formation of prostaglandins and thromboxane in plasma samples	Add indomethacin to samples during collection. Keep samples on ice whenever possible. Measure downstream metabolites rather than primary eicosanoids.
During sample collection, storage, and processing	Oxidation of bioactive lipids	Exogenous formation of oxidatively damaged lipids, such as isoprostanes	Add BHT to samples during collection. Keep samples on ice whenever possible. Store samples at -80°C.
During sample processing	AEBSF added to cells or tissues during processing	AEBSF inhibits acetylcholinesterase, which is used as a tracer in many of Cayman's ELISAs.	Use a different serine protease inhibitor.

Minimizing exogenous formation of eicosanoids

Eicosanoids and other bioactive lipids are often formed exogenously during the collection and processing of samples. Inhibitors of the enzymes responsible for eicosanoid formation, or those that inhibit oxidation can be added during sample collection and processing to attenuate the formation of these unwanted products. A list of suggested inhibitors is below.

Compound	Inhibitor of:	Why?
Indomethacin	Cyclooxygenases	Inhibits formation of exogenous prostaglandins and thromboxanes.
Nordihydroguaiaretic Acid	Lipoxygenases	Inhibits exogenous formation of leukotrienes and HETEs.
BHT	Oxidation	Attenuates oxidation of lipids, which could otherwise result in artificially elevated levels of compounds such as isoprostanes.

Ready-to-use preservation vials are available for storing blood samples

Sampling tubes with Indomethacin– designed to preserve 1 ml samples for future analysis of prostaglandins and thromboxanes

Sampling tubes with BHT – designed to preserve 1 ml samples for future analysis of isoprostanes

Sampling tubes with BHT and Indomethacin – designed to preserve 1 ml samples for future analysis of prostaglandins, thromboxanes, and isoprostanes

Storage

Cayman recommends storing biological samples to be used for measurement of bioactive lipids at -80°C. This is of particular importance when measuring oxidative damage (i.e., isoprostanes), as lipid oxidation continues at -20°C.

Sample preparation/purification

The goal of sample prep and purification is to remove substances that interfere with accurate quantification of a bioactive lipid in your sample. When bioactive lipid levels are high, purification may not be necessary as interfering substances will be diluted out. When bioactive lipid levels are lower, more stringent purification will be required. Some general techniques are listed below.

Protein precipitation – Protein precipitation is most commonly used with highly proteinaceous samples, such as cell lysates or tissues, prior to performing solid phase extraction.
Liquid:liquid extraction – This technique takes advantage of the organic solubility of bioactive lipids, separating them from compounds that are only soluble in aqueous solution.
Solid phase extraction (SPE) – C18-SPE is commonly used in a manner similar to liquid:liquid extraction to separate bioactive lipids from compounds that are soluble only in aqueous solution.
Immuno-affinity sorbent – Cayman has immuno-affinity sorbents that will specifically recognize individual bioactive lipids (e.g., 8-isoprostane ). Purification of a sample using an immuno-affinity sorbent is the most analyte-specific method for purification.

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