Anal. Chem. 1996, 68, 4101-4106
Simultaneous Determination of Cross-Reactive Leukotrienes in Biological Matrices Using On-line Liquid Chromatography Immunochemical Detection A. J. Oosterkamp,† H. Irth,*,† L. Heintz,‡ G. Marko-Varga,‡ U. R. Tjaden,† and J. van der Greef†
Leiden/Amsterdam Center for Drug Research, Division of Analytical Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, and Bioanalytical Chemistry, Pre-Clinical Research & Development, Astra Draco AB, P.O. Box 34, S-221 00 Lund, Sweden
Sulfidopeptide leukotrienes, which are important biomarkers for several diseases, are commonly measured by microtiter plate immunoassays. These immunoassays, however, cannot distinguish between several structurally similar leukotrienes and their cross-reactive metabolites and, therefore, need extensive sample handling and fractionation by means of liquid chromatography (LC). This paper describes the development and automation of a continuous-flow immunochemical detection (ICD) system and its subsequent on-line coupling to LC. The online LC-ICD system based on fluorescence-labeled leukotriene E4 (LTE4) was used to determine sulfidopeptide leukotrienes and their cross-reactive metabolites in a single run. Furthermore, biological matrices, e.g., urine and human cell extracts, were analyzed, the only sample pretreatment being on-line solid-phase extraction (SPE) on a novel RP-C4 restricted-access support. The determination limit of LTE4 in urine was 0.2 ng/mL (800 fmol; injection volume, 2000 µL; signal-to-noise ratio, 10). The system was linear from 0.2 to 1.0 ng/mL LTE4. Using nonlinear curve-fitting, the range could be expanded to 2.5 ng/mL. It is shown that, besides quantitation of known analytes, on-line LC-ICD is useful in the discovery of cross-reactive LTE4 metabolites. Sulfidopeptide leukotrienes (LTC4, LTD4, and LTE4, Figure 1) are synthesized through the lipoxygenase pathway and play an important role in pathological events of inflammation and allergic reactions.1 They are potent bronchoconstrictor substances. Relevant amounts of LTE4 are produced in the human lung after immunochemical challenge in vitro, suggesting a potent role for sulfidopeptide leukotrienes in the pathogenesis of atopic asthma. Leukotrienes have also been detected in vivo in bronchoalveolar lavages obtained after endobronchial stimulation with antigens, as well as in tracheal aspirates from patients affected by acute respiratory distress (ARDS) or by extensive burn injury of the respiratory tract.2 †
Leiden University. Astra Draco AB. (1) Samuelsson, B.; Dahle´n, S. E.; Lindgren, J.-Å.; Rouzer, C. A.; Serhan, C. N. Science 1987, 237, 1171-1176. (2) Sala, A.; Armetti, L.; Piva, A.; Folco, G. Prostaglandins 1994, 47, 281-292. ‡
S0003-2700(96)00250-8 CCC: $12.00
© 1996 American Chemical Society
Figure 1. Biosynthesis of leukotrienes.
LTC4 shows an extensive metabolism after its biosynthesis from arachidonic acid. LTC4 is converted to LTE4, which in its turn is believed to be rapidly excreted in the bloodstream and excreted in the urine directly or after metabolization, e.g., by various cycles of ω- or β-oxidation, N-acetylation, or sulfoxidation.3,4 Unmetabolized LTE4 is, on the other hand, described as a useful (3) Huber, M.; Mu ¨ ller, J.; Leier, I.; Jedlitschky, G.; Ball, H. A.; Moore, K. P.; Taylor, G. W.; Williams, R.; Keppler, D. Eur. J. Biochem. 1990, 194, 309315. (4) Jedlitschky, G.; Leier, I.; Huber, M.; Mayer, D.; Keppler, D. Arch. Biochem. Biophys. 1990, 282, 333-339.
Analytical Chemistry, Vol. 68, No. 23, December 1, 1996 4101
urinary marker for the systemic sulfidopeptide leukotriene formation.2,5 The preferred analytical methods for the determination of leukotriene concentrations in biological material are highperformance liquid chromatography (HPLC) and immunoassays. HPLC methods are advantageous if several leukotrienes have to be determined at the same time.6 Moreover, HPLC methods are easy to automate. In some cases, however, the extensive sample pretreatment and low sensitivity outweigh these advantages. Furthermore, the results of HPLC-UV methods can be erroneous due to unpure peaks.7 Immunoassays represent very sensitive assays where often no cleanup of samples such as culture supernatants, plasma, serum, or urine, is needed. For sulfidopeptide leukotrienes, only a few polyclonal and monoclonal antibodies are described which exhibit sufficient selectivity in the immunoassay.8,9 However, these antibodies still show extensive cross-reactivity to several leukotriene metabolites. Therefore, in many cases, immunoassays for leukotrienes are coupled to fractionation steps using solid-phase extraction (SPE) and HPLC to improve selectivity.2,10,11 In our laboratory, continuous-flow immunochemical detection (ICD) methods have been developed which can be coupled online to reversed-phase HPLC. On-line coupling combines the separation power of HPLC with the sensitivity and selectivity of immunoassays and overcomes the tedious fractionation steps which are necessary in the off-line coupling of LC with ICD.12 Both fluorescence-labeled antibody-13,14 and fluorescence-labeled antigenbased15 ICD systems have been developed. Due to highly selective ICD, sample pretreatment in LC can be drastically reduced. Simple on-line deproteination and simultaneous preconcentration on a restricted access precolumn are sufficient to measure selectively the compounds of interest at nanomole per liter concentrations.14 Also, receptor-ligand interactions have successfully been implemented in on-line biochemical detection setups.16 The present paper describes the development of a continuousflow ICD system based on fluorescence-labeled antigens and the subsequent on-line coupling to LC for the determination of leukotrienes. In the ICD system, a monoclonal antibody against LTD4 which shows high cross-reactivity for sulfidopeptide leukotrienes is used8 (for cross-reactivity data, see Table 1). The developed method is used for the simultaneous determination of LTC4, LTD4, and LTE4 in biological samples, e.g., cell extracts (5) Celardo, A.; Dell’Elba, G.; Eltantawy, Z. M.; Evangelista, V.; Cerletti, C. J. Chromatogr. B. 1994, 658, 261-269. (6) Yu, W.; Powell, W. S. Anal. Biochem. 1995, 226, 241-251. (7) Heintz, L.; Osterlins, E.; Alkner, U.; Marko-Varga, G. Submitted for publication. (8) Reinke, M.; Hoppe. U.; Ro ¨der, T.; Bestmann, H.-J.; Mo ¨llerhauer, J.; Brune, K. Biochim. Biophys. Acta 1991, 1081, 274-278. (9) Hayes, E. C. Methods Enzymol. 1990, 187, 116-124. (10) Gelpi, E.; Ramis, I.; Hotter, G.; Bioque, G.; Bulbena, O.; Rosello´, J. J. Chromatogr. 1989, 492, 223-250. (11) Nicoll-Griffith, D.; Zamboni, R.; Rasmussen, J. B.; Ethier, D.; Charleson, S.; Tagari, P. J. Chromatogr. 1990, 526, 341. (12) Irth, H.; Oosterkamp, A. J.; Tjaden, U. R.; van der Greef, J. Trends Anal. Chem. 1995, 14, 355-361. (13) Irth, H.; Oosterkamp, A. J.; van der Welle, W.; Tjaden U. R.; van der Greef, J. J. Chromatogr. 1993, 633, 65-72. (14) Oosterkamp, A. J.; Irth, H.; Beth, M.; Unger, K. K.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. B 1994, 653, 55-61. (15) Oosterkamp, A. J.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1994, 66, 4295-4301. (16) Oosterkamp, A. J.; Villaverde Herraı´z, M. T.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Chem. 1996, 68, 1201-1206.
4102 Analytical Chemistry, Vol. 68, No. 23, December 1, 1996
Table 1. Characteristics of the Anti-LTD4 Antibody8 eicosanoids
cross-reactivity (IC50) (%)
leukotriene C4 leukotriene D4 leukotriene E4 N-Acetylleukotriene D4 leukotriene B4 tromboxane B2
95.7 100.0 88.7 89.7
