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Spectrophotometric determination of neutrophil cytochrome b 558 of chronic granulomatous disease
Author(s) -
Kiryu Chika,
Kaneda Mizuho,
Shiraishi Takuo,
Tsuda Masahiko,
Inana Ichiro,
Sakiyama Takeshi,
Fujinaga Toru,
Nishida Akira,
Kakinuma Katsuko
Publication year - 1998
Publication title -
pediatrics international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.49
H-Index - 63
eISSN - 1442-200X
pISSN - 1328-8067
DOI - 10.1111/j.1442-200x.1998.tb01913.x
Subject(s) - chronic granulomatous disease , heme , oxidase test , medicine , nadph oxidase , hemoglobin , cytochrome b , cytochrome , cytochrome c oxidase , spectrophotometry , cytochrome c , microbiology and biotechnology , biochemistry , immunology , enzyme , chromatography , chemistry , mitochondrion , gene , biology , oxidative stress , mitochondrial dna
Background: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe 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 ol 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 foi 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 α and β bands and differentiated as d[ΔA]/dΛ, (Λ= wavelength) to obtain further evidence for the defects of the cyt b 558 heme spectrum. The interference from CO‐insensitive met Hb was eliminated by subtracting the absorption peak at the Soret (γ) band of the contaminating met Hb, which was estimated from the CO‐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 in10–20 mL of blood even in the presence ol contaminating Hb.