Spectrophotometric determination of neutrophil cytochrome b₅₅₈ of chronic granulomatous disease

Background: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severs. recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (O-2(-)) by the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b(558) (cyt b(558)), which is one of the oxidase components. Diagnosis of CGD has been performed by the assay of the O-2(-) forming activity, immunological determination of defects in the oxidase components, and or spectrophotometry of cyt b(558). However, spectrophotometric analysis of the b-type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b(558). This report presents an accurate method for the spectrophotometric analysis of cyt b(558) in a small amount of CGD neutrophils that were treated with CO gas in a safe procedure instead of the previously reported CO-bubbling method. Methods and Results: The difference of the reduced minus oxidized cyt b(558) spectrum was measured under no interference from oxy Hb at 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-treated 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.