TY - JOUR

T1 - A specific asay for lukotriene B4 in human whole blood

AU - Fogh, Jytte

AU - Poulsen, Lars K.

AU - Bisgaard, Hans

PY - 1992/12

Y1 - 1992/12

N2 - Leukotrienes (LTs) are potent mediators of inflammatory and allergic responses, and are present in biological fluids in minute amounts, that is, in the picogram range. The aim of this study was to develop and validate a method for determination of LTB4 synthesized in vitro in human whole blood. Heparinized blood was stimulated with calcium-ionophore A23187 at 37°C. After 30 min cells were separated by centrifugation. LTB4 was analyzed by radioimmunoassay (RIA). When sample preparation was restricted to protein precipitation with acetone, interference was demonstrated by lack of parallelism between standard and sample dilution curves. Purification was, therefore, extended by combinations of the following steps: 1) protein precipitation, 2) lipid extractions, and 3) high-performance liquid chromatography (HPLC). One of two commercially available LTB4 standards was found to contain multiple components, several of which were immunoreactive in RIA. Even for the standard containing pure LTB4, interference was demonstrated by lack of parallelism between sample and standard dilution curves. Testing eight combinations of varying purification steps, we found that only a three-step purification procedure, including 1) solid-phase extraction, 2) protein precipitation at -20°C, and 3) HPLC, was able to eliminate interference in RIA. Using this procedure, the recovery was 78%. Stimulation of whole blood from normal subjects with calcium-ionophore showed optimal LTB4 production at 10 μM ionophore, yielding 6.6 ng LTB4/mL blood.

AB - Leukotrienes (LTs) are potent mediators of inflammatory and allergic responses, and are present in biological fluids in minute amounts, that is, in the picogram range. The aim of this study was to develop and validate a method for determination of LTB4 synthesized in vitro in human whole blood. Heparinized blood was stimulated with calcium-ionophore A23187 at 37°C. After 30 min cells were separated by centrifugation. LTB4 was analyzed by radioimmunoassay (RIA). When sample preparation was restricted to protein precipitation with acetone, interference was demonstrated by lack of parallelism between standard and sample dilution curves. Purification was, therefore, extended by combinations of the following steps: 1) protein precipitation, 2) lipid extractions, and 3) high-performance liquid chromatography (HPLC). One of two commercially available LTB4 standards was found to contain multiple components, several of which were immunoreactive in RIA. Even for the standard containing pure LTB4, interference was demonstrated by lack of parallelism between sample and standard dilution curves. Testing eight combinations of varying purification steps, we found that only a three-step purification procedure, including 1) solid-phase extraction, 2) protein precipitation at -20°C, and 3) HPLC, was able to eliminate interference in RIA. Using this procedure, the recovery was 78%. Stimulation of whole blood from normal subjects with calcium-ionophore showed optimal LTB4 production at 10 μM ionophore, yielding 6.6 ng LTB4/mL blood.

KW - High-performance liquid chromatography

KW - Leukotriene B

KW - Radioimmunoassay

KW - Whole blood

UR - http://www.scopus.com/inward/record.url?scp=0027105056&partnerID=8YFLogxK

U2 - 10.1016/1056-8719(92)90002-I

DO - 10.1016/1056-8719(92)90002-I

M3 - Journal article

C2 - 1338371

AN - SCOPUS:0027105056

VL - 28

SP - 185

EP - 190

JO - Journal of Pharmacological and Toxicological Methods

JF - Journal of Pharmacological and Toxicological Methods

SN - 1056-8719

IS - 4

ER -