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Pharmacological Research, Vol . 26, Supplement 2, 1992

EICOSANOIDS IN ALLERGIC REACTIONS: QUANTITATIVEDETERMINATION BY ENZYME IMMUNOASSAY

S. Nicosia, T. Vigano', G .C. FolcoInstitute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133

Milano, Italy

Key words: Leukotrienes, PGD 2 , enzyme, immunoassay, asthma .

Prostaglandins (PG) and leukotrienes are lipid mediators implicated in

various inflammatory or allergic reactions (1) . They possess a broad spectrum of

biological activities and are present in biological fluids in extremely low

concentrations; thus, highly sensitive methods are required for their analysis . We

have applied an enzyme immunoassay (EIA) to the quantitative determination of

PGD2 and leukotrienes in allergic reactions . In human lung fragments under

conditions of non immunologic stimulation (ionophore A-23187 1 µM) predominant

formation of sulfidopeptide leukotrienes and LTB 4 is observed (LTD4 0.33 ± 0.30 ;

LTE4 0.61 ± 0 .45; LTB4 0 .32 ± 0 .22 nmoles/g tissue) . Following immunologic

challenge (anti-human IgE 70 µg/ml) approximately 70 per cent of the precursor

arachidonic acid is transformed to PGD 2 (1 .27 ± 0.71 nmoles/g tissue), while LTD4

(0.21 ± 0.16 nmoles/g tissue) and LTE4 (0 .28 ± 0.21 nmoles/g tissue) together

comprise about 25 percent of the overall metabolism ; the amounts of LTC4 and

LTB4 are negligible .

Pretreatment of the pulmonary tissue with 1 .5 x 10 -5 M indomethacin inhibits

cyclo-oxygenase activity (expressed as PGD 2 levels) by more than 90% . No

redistribution of precursor metabolism to the sulfidopeptide leukotrienes takes

place .

Purification of samples with HPLC (2) is the first step in the analysis by EIA of

leukotrienes present in the lavage fluid obtained from an asthmatic patient

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© 1992 The Italian Pharmacological Society

Pharmacological Research, Vol . 26, Supplement 2, 1992

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challenged intrabronchially with Dermatophagoides pteronyssinus . The specific

challenge causes oedema of the bronchial mucosa and narrowing of the bronchial

diameter as observed through the bronchoscope 2-3 min after stimulus . This is

associated with biochemical events as evidentiated with EIA of sulfidopeptide

leukotrienes in HPLC-derived fractions corresponding to exact retention time of

leukotrienes. The amount of immunoreactive LTE4 is more than two-folds at 5 min

(510 ± 130 pg/ml) compared to controls (p<0 .05) .

Previous addition of radioactive leukotrienes to the sample allows an

estimation of immunoreactive material considering the recovery of the extraction

and purification . This demonstration confirm the critical role of eicosanoids in

allergic asthma ; moreover the results obtained by EIA confirm the role of this

immunoassay as a substitute of radioimmunoassay for quantitative determination of

these compounds .

REFERENCES

1) Lewis R.A ., Austen K.F. The biologically active leukotrienes . J . Invest . 1984 ; 73 :

889-897 .

2) Powell W.S. Rapid extraction of arachidonic acid metabolites from biological

samples using actadodecylsill silica. Methods Enzymol . 1982; 86: 467-477 .

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