Effect of physical and chemical methods of homogenization on inflammatory mediators in sputum of asthma patients

Background: Dithiothreitol (DTT), which is used for sputum homogenization, may split S-S bonds of the bronchial mucins as well as other proteins and, thus, may have a detrimental effect on inflammatory mediators that are pres ent in sputum. Objective: To evaluate the effects of physical sputum homogenization, using ultrasonic and chemical (ie, DTT) means, on the concentrations of eosinoph il cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidas e (EPO), and myeloperoxidase (MPO) in the sputum of patients with asthma. Methods: The collection of sputum samples from nine patients with asthma wa s induced by their inhaling a sterile 3% saline solution for 10 min from an ultrasonic nebulizer. One half of the sputum sample was homogenized by ult rasound, and the other half was liquefied by DTT. The supernatant of the ul trasonically homogenized specimen was divided into the following three port ions: (1) immediately frozen; (2) stored for 15 min at 37 degreesC; and (3) additionally treated with DTT. The supernatant of the sputum sample that w as Liquefied by DTT was divided into the following two portions: (1) immedi ately frozen; and (2) additionally subjected to ultrasound. The concentrati ons of ECP, EPO, EPX, and MPO in the sputum samples were measured using imm unoassays. Results: Statistically significant differences were found between the ultra sonically homogenized specimens that had been either processed immediately or stored at 37 degreesC and those treated by DTT, but only for concentrati ons of EPO and MPO (p < 0.005). No effect of temperature on the mediators i n the ultrasonically homogenized specimens could be detected. Ultrasonic ho mogenization had no influence on the mediators in the samples liquefied by DTT. However, the addition of DTT to the cell-free supernatant of the ultra sonically homogenized sputum samples caused a significant fall in measured EPO and MPO concentrations. Conclusions: The sputum processing by DTT caused a statistically significan t fall in EPO and MPO concentrations but did not significantly influence th e measured concentrations of ECP and EPX.