Measurements of Arachidonic Acid Metabolites Derived from the Lipoxygenase Pathways by High-Pressure Liquid Chromatography
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|
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 |
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|
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 |
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|
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 |
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|
Amersham |
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|
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 |
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|
Lipoxygenase |
1. HPLC |
PGB2 |
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|
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 |
