Spectrophotometric determination of neutrophil cytochrome b558 of chronic granulomatous disease.
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 (O2-) by the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b558 (cyt b558), which is one of the oxidase components. Diagnosis of CGD has been performed by the assay of the O2- forming activity, immunological determination of defects in the oxidase components, and or spectrophotometry of cyt b558. 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 b558. This report presents an accurate method for the spectrophotometric analysis of cyt b558 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.


RESULTS
The difference of the reduced minus oxidized cyt b558 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 b558 heme spectrum. The interference from CO-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 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 b558 with a small amount of CGD neutrophils in 10-20 mL of blood even in the presence of contaminating Hb.
Kiryu
C
C
Biophotonics Information Laboratories, Yamagata Advanced Technology Research Center, Japan.
Kaneda
M
M
Shiraishi
T
T
Tsuda
M
M
Inana
I
I
Sakiyama
T
T
Fujinaga
T
T
Nishida
A
A
Kakinuma
K
K
D003573:Cytochrome b Group / Q000097:blood*; D005260:Female; D006105:Granulomatous Disease, Chronic / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D007223:Infant; D008297:Male; D008706:Methemoglobin / Q000032:analysis; D009504:Neutrophils / Q000378:metabolism; D010108:Oxyhemoglobins / Q000032:analysis; D009195:Peroxidase / Q000032:analysis; D013053:Spectrophotometry / Q000379:methods*
D003573:Cytochrome b Group; D010108:Oxyhemoglobins; D008706:Methemoglobin; D009195:Peroxidase