A group of 20 patients with proven EIA participated in the study. There were 13 males and seven females ranging in age from 10 to 43 years (mean age 23). All patients underwent an initial measurement of pulmonary function by Spirograph, Jaeger Compactransfer, Germany, in which all of them showed a reduction of at least 15% in their forced expiratory volume in 1 s (FEV1) after a 7-min run on a motorized Quinton treadmill followed by an 8-min rest. The workload was the same for each patient exercising up to a submaximal effort of 80% of the theoretical maximal heart rate, as indicated by an Omeda 3700e pulse oxymeter. All subsequent exercise testing was performed under the same ambient conditions for all patients and with the same workload that produced EIA at the baseline evaluation. Pulmon-ary functions were measured at the same time of the day with similar ambient conditions to avoid any variability. Exercise testing as described above was performed before and after a 7-day supplementation period of 30 mg lycopene or placebo, administered randomly in a double-blind fashion. Each patient underwent this protocol twice, receiving either lycopene or placebo, with a 4-week interval between each protocol to allow washout. Twelve hours before evaluation of the pulmonary functions, all patients discontinued their regular medications, which consisted of antihistamines, short-action bronchodilators, and inhaled steroids. The dose of inhaled steroids was constant for several weeks before each challenge. Challenge was not done within several weeks of a respiratory tract infection. Patients avoided allergen exposure for several weeks before each challenge.
The lycopene preparation used was LYC-O-MATO, an oleoresin product manufactured by LycoRed Natural Products Industries Ltd, Beer Sheva 84102, Israel. LYC-O-MATO contains 6% lycopene, 1.6% tocopherols, 1% phytoene and phytofluene, 0.25%β-carotene, and other minor phytochemicals extracted from tomato. LYC-O-MATO was introduced in the form of soft capsules of 15 mg (6%) lycopene.
Lycopene, carotenoids, retinol, and tocopherol serum concentrations were measured on blood samples collected after supplementation of placebo or lycopene. Blood samples were taken after overnight fasting from 7 to 9 am. Blood was separated for routine blood tests and for vitamin analysis. Serum was obtained by centrifugation at 3000g at 5°C and stored at −70°C under nitrogen until analysis, usually within 7 days. Ethanol (2.5 ml) was added to 1 ml serum, and after vigorous mixing the lipophilic fraction was extracted with 5 ml n-hexane through phase separation and centrifugation at 2000 g at 5°C for 5 min. The upper phase was removed and the water-ethanol phase extracted a second time with 3 ml n-hexane. The two hexane extracts were combined, evaporated to 1 ml for spectrophotometric quantitative analysis, and then to dryness by a stream of nitrogen. The dried residue was dissolved in 100 µl methylene chloride before being injected into the HPLC system. This was a Waters HPLC system (Millipore, Marlborough, MA, USA). The system included pumps 501 and 510 and a Waters 996 photodiode array detector attached to Waters Millennium 2010, Chromatography Manager, Version 2.10, run on an IBM-compatible computer connected to an HP DeskJet 1200 plotter (Hewlett Packard, Avondale, PA, USA). The column was a Vydac 201 TP54 stainless steel column of 25 cm x 4.6 mm (internal diameter) packed with C-18 reversed-phase material with particle size of 5 µm and a pore size of 30 nm (The Separation Group, Hysperia, CA, USA). The column was maintained at 30±0.2°C in an HPLC Column 7955 Heater/Chillier (Jones Chromatography, Glamorgan, UK). The column was protected by a 5-cm C-18 ODS guard column (Shimadzu, Kyoto, Japan) and with a small preguard column, a Guard-Pak, inserted with a C-18 µBondapak cartridge (Waters Chromato-graphy, Milford, PA, USA). Elution was performed at 30±0.2°C with an isocratic solvent, HPLC grade methanol:acetonitrile (9:1 by vol), at a constant flow of 1.0 ml/min, which is a well-documented system for distinguishing the different carotenoids and their isomers ( 12, 28, 30). The mobile phase was flushed with nitrogen to avoid air gassing in the solvents. Samples were injected with a 7725i syringe-loading sample injector fitted with a 5-μl loop (Rheodyne, Inc., Cotati, CA, USA). Peak responses were measured and assessed at their maximum wavelengths with the photodiode array and detected by the Millennium 3-D “Max” absorption, as described previously. Peak responses of carotenoids were measured at 450 nm. Excel (Microsoft, USA) was used for quantification of the HPLC data. The standards, lutein, β-apo-8′-caro-tenal, β-cryptoxanthin, all-trans-β-carotene, α-carotene, α-tocopherol, and lycopene, were purchased from Sigma (St Louis, MO, USA). All standards were kept at −70°C under nitrogen, and dried by a stream of nitrogen before being analyzed and injected into the HPLC system in methylene chloride. The concentration of the standards was determined by spectral measurement and calculated with the appropriate extinction coefficients in ethanol. β-apo-8′-carotenal was used as internal standard in all runs. An amount of 2.5 µg of standard were added to the serum with THF:MeOH, and then the internal standard was extracted as described above for injection into the HPLC at 125 ng/5 µl. We did not note any loss in the quantity of the internal standard along the extraction process; nevertheless, we ensured linearity by using three concentrations of the standard.