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 present in sputum.


OBJECTIVE
To evaluate the effects of physical sputum homogenization, using ultrasonic and chemical (ie, DTT) means, on the concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO), and myeloperoxidase (MPO) in the sputum of patients with asthma.


METHODS
The collection of sputum samples from nine patients with asthma was 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 ultrasound, and the other half was liquefied by DTT. The supernatant of the ultrasonically homogenized specimen was divided into the following three portions: (1) immediately frozen; (2) stored for 15 min at 37 degrees C; and (3) additionally treated with DTT. The supernatant of the sputum sample that was liquefied by DTT was divided into the following two portions: (1) immediately frozen; and (2) additionally subjected to ultrasound. The concentrations of ECP, EPO, EPX, and MPO in the sputum samples were measured using immunoassays.


RESULTS
Statistically significant differences were found between the ultrasonically homogenized specimens that had been either processed immediately or stored at 37 degrees C and those treated by DTT, but only for concentrations of EPO and MPO (p < 0.005). No effect of temperature on the mediators in the ultrasonically homogenized specimens could be detected. Ultrasonic homogenization had no influence on the mediators in the samples liquefied by DTT. However, the addition of DTT to the cell-free supernatant of the ultrasonically homogenized sputum samples caused a significant fall in measured EPO and MPO concentrations.


CONCLUSIONS
The sputum processing by DTT caused a statistically significant fall in EPO and MPO concentrations but did not significantly influence the measured concentrations of ECP and EPX.
Grebski
E
E
Pulmonary Division, Department of Internal Medicine, University of Zurich, Switzerland.
Peterson
C
C
Medici
T C
TC
D000328:Adult; D000368:Aged; D001249:Asthma / Q000276:immunology*; D001798:Blood Proteins / Q000032:analysis*; D019963:Clinical Chemistry Tests / Q000379:methods; D004229:Dithiothreitol / Q000494:pharmacology*; D047091:Eosinophil Granule Proteins; D047209:Eosinophil Peroxidase; D047208:Eosinophil-Derived Neurotoxin; D004804:Eosinophils / Q000201:enzymology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009195:Peroxidase / Q000032:analysis*; D010544:Peroxidases / Q000032:analysis*; D055585:Physical Phenomena; D010825:Physics; D012260:Ribonucleases / Q000032:analysis*; D013183:Sputum / Q000737:chemistry*; D014465:Ultrasonics
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D047209:Eosinophil Peroxidase; D010544:Peroxidases; D009195:Peroxidase; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases; D004229:Dithiothreitol