Measurements of Arachidonic Acid Metabolites Derived from the Lipoxygenase Pathways by High-Pressure
We have summarized in Chapter 16 the recent advances in the mechanisms of regulation of leukotriene and prostaglandin biosynthesis and their pathways of synthesis. This chapter focuses on the measurement of the leukotrienes and other LOX products by high-pressure liquid chromatography (HPLC). At this point, we also present a complete summary in Table 1 comparing the various approaches used for the measurements of arachidonic acid metabolites derived from the prostaglandin endoperoxide synthase and the lipoxygenase pathways, as presented in Chapters 16 –18 . Table 1 Characteristics of the Various Assays for Arachidonic Acid Metabolites
Mediator | Assays | Sources | Mediators | Tracers | Sensitivity (pg/ml) | Range (pg/ml) | Timeframe ft1|Will vary depending on the temperature of incubation (e.g., 2–8�C, 25�C , or 37�C) | No of samples (lubes vs wells) | Advantages | Limitations |
|---|---|---|---|---|---|---|---|---|---|---|
Prostanoids | 1. RIA (charcoal) | Sigma/Amersham | PGE2 | [3 H] | 15pg/tube | 80–80 000 | ovemight | - longer procedure | ||
6-keto-PGF1α | [3 H] | 10pg/tube | 80–20 000 | ovemight | 120/btle antiserum | - less expensive | - not suitable for | |||
PGE2α | [3 H] | 5pg/tube | 90–10 000 | ovemight | 6000/btle tracer | biological fluids | ||||
TxB)2 | [3 H] | 4pg/tube | 20–5 000 | ovemight | (unless extracted) | |||||
And some more | - radioactivity | |||||||||
Amersham | PGD2 | [3 H] | 20 | 30–2 000 | ovemight | 100 | ||||
6-keto-PGF1α | [3 H] | 140 | 140–5 000 | 2 days | 100 | - suitable urine/plasma | ||||
PGF2α | [3 H] | 30 | 30–3 000 | ovemight | 100 | - need pre-purification | ||||
TxB2 | [3 H] | 20 | 50–3 000 | 2 days | 100 | - suitable plasma | ||||
2. RIA + Amerlex-M ft2|With the magnetic polymer beads, simple magnetic eparation, eliminating centrifugation | Amersham | PGF2 | [125 H] | 8 | 12.5–1600 | 2.5 h | 100 | -suitable urine/plasma | -limited tracer's life | |
6-keto-PGF1α | [125 H] | <20 | 30–4 000 | ovemight | 100 | -suitable plasma | -more radioactivity | |||
TxB2 | [125 H] | <20 | 30–4 000 | ovemight | 100 | -suitable plasma | ||||
3. RIA + SPA ft3| Scintillation proximity assays do not necessitate to separate bound from free ligand, more automation, increase throughout, greater productivity, no solvents, vials, and so on (see Amersham). | Amersham | PGF2 | [125 H] | 10 | 12.6–1 600 | ovemight | 100 | - suitable urine/plasma | - radioactivity | |
6-keto-PGF1α | [125 H] | 140 | 140–5 000 | ovemight | 100 | - suitable urine/plasma | ||||
TxB2 | [3 H] | 10 | 50–3 000 | 1 day | 100 | - suitable plasma | ||||
4. EIA ft4|Rapid, nonisotopic, one plate, less manipulation, faster reading with EIA plate reader (vs γ-counter), no centrifugation. | Cayman | PGF2 | AChE | 29 | 7.8–1000 | ovemight | 96/plate | - monoclonal | - need pre-purification | |
6-keto-PGF1α | AChE | 23.6 | 3.9–500 | ovemight | 96/plate | |||||
PGF2α | AChE | 24 | 15.6–2000 | ovemight | 96/plate | |||||
TxB2 | AChE | 13.3 | 7.8–1000 | ovemight | 96/plate | |||||
And many more | - non isotopic | |||||||||
Amersham | ||||||||||
PGF2 | HRP | 16 | 20–640 | 5h | 96/plate | - fast assay | ||||
6-keto-PGF1α | HRP | 3 | 10–1280 | 2h | 96/plate | - suitable urine/plasma | ||||
TxB2 | HRP | 3.6 | 10–1280 | 2h | 96/plate | - suitable urine/plasma | ||||
Leukotrienes | 1. RIA (charcoal) | Sigma/Amersham | LTB4 LTC4 /D4 /E4 | [3 H]] [3 H]] | ||||||
Amersham | LTB4 | [3 H] | 15 | 16–2 000 | 3– h or ovemight | 125 | - need pre-purification | |||
LTC4 | [3 H]] | 88 | 80–5 000 | Ovemighl | 100 | - suitable urine/plasma | ||||
LTC4 /D4 /E4 | [3 H]] | 50 | 125–8 000 | 1 h or ovemight | 125 | - need pre-purification | ||||
2. RIA + SPA | LTB4 | [3 H]] | 62 | 62–2 000 | ovemight | 500 | - need pre-purification | |||
3. EIA | Cayman | LTB4 | AChE | 2.23 | 3.9–500 | ovemight | 96/plate | |||
LTC4 | AChE | 25 | ovemight | 96/plate | ||||||
LTE4 | AChE | 28.7 | ovemight | 96/plate | ||||||
LTC4/ D4 /E4 | AChE | 11.8 | ovemight | 96/plate | -fast assay | |||||
Amersham | LTB4 | HRP | 6 | 62–800 | 3.5 h | 96/plate | -suitable plasma plasma | |||
LTC4 /D4 /E4 | HRP | 10 | 15–960 | 5.5 h | 96/plate | - suitable plasma | ||||
Isoprostanes | 1. EIA | Cayman | 8-iso PGF2α | AChE | 6 | ovemight | 96/plate | see above EIA | see above EIA | |
-analysis of several products at one time | -expensive eajuipmenl required | |||||||||
Lipoxygenase | 1. HPLC | PGB2 | ||||||||
products | LTB4 LTC4 /D4 /E4 HETEs Lipoxins Upo Jtins | 1–5ng(LTs-LXs) 5– ng(HETEs) | 40 to 50 min per sample | -minimal sample preparation procedures -internal standards - high level selectivity | - limit of detection ≥1 ng |
1 Will vary depending on the temperature (e.g., 2–8�C, 25�C or 37�C). 2 With the magnetic polumner beads, simple magnetic sepration, elimiminating centrifugation. 3 Scintillation proximity assays do not assays do not necessitate to separate bound from free ligand, more automation, increase throughout, greater productivity, no solvents, vials, and so on. (see Amersham). 4 Rapid, nonisotopic, one plate, less manipulation, faster reading with EIA plate reader (vx γ-counter), no centrifugation.
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