SPECTROPHOTOMETRIC DETERMINATION OF NEUTROPHIL CYTOCHROME B(538) OF CHRONIC GRANULOMATOUS-DISEASE

Background: Chronic granulomatous disease (CGD) is an inherited diseas e characterized clinically by severs. recurrent bacterial infections f rom infancy. This disease is a disorder of the formation of superoxide (O-2(-)) by the neutrophil NADPH oxidase system, mostly due to defect s in cytochrome b(558) (cyt b(558)), which is one of the oxidase compo nents. Diagnosis of CGD has been performed by the assay of the O-2(-) forming activity, immunological determination of defects in the oxidas e components, and or spectrophotometry of cyt b(558). However, spectro photometric analysis of the b-type heme is difficult with small amount s of blood from infant CGD patients, as the limited amounts of neutrop hils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b(558). This report pres ents an accurate method for the spectrophotometric analysis of cyt b(5 58) in a small amount of CGD neutrophils that were treated with CO gas in a safe procedure instead of the previously reported CO-bubbling me thod. Methods and Results: The difference of the reduced minus oxidize d cyt b(558) spectrum was measured under no interference from oxy Hb a t the alpha and beta bands and differentiated as d[Delta A]/d lambda ( lambda = wavelength) to obtain further evidence for the defects of the cyt b(558) heme spectrum. The interference from GO-insensitive met Hb was eliminated by subtracting the absorption peak at the Soret (gamma ) band of the contaminating met Hb, which was estimated from the GO-tr eated and untreated spectra of the same, hemolyzed sample. Conclusions : This spectrophotometric method is feasible for the determination of abnormality and heme content of cyt b(558) with a small amount of CGD neutrophils in 10-20 mt of blood even in the presence of contaminating Hb.