New ultra-micro high-performance liquid chromatographic method for determining the gamma chain composition of hemoglobin F in normal adults.
The difficulty in isolating the minute quantity of Hb F (< 1%) present in the red blood cells of normal adults greatly complicates the determination of its gamma chain composition. We have developed a rapid ultra-micro high-performance liquid chromatographic (HPLC) method and used it to analyze the gamma chain composition of Hb F in 47 adults with Hb F levels between 0.1-3.4%. The method involves the isolation of Hb from as little as 50 microliters of whole blood on an analytical size cation-exchange HPLC column, followed by concentration in a Centricon micro concentrator unit and by reversed-phase HPLC analysis. The entire procedure can be completed in one day and 3-4 analyses can be made simultaneously. We reanalyzed the blood samples from 22 subjects with known beta-globin gene cluster haplotypes, and confirmed the association of high, low, and very low G gamma levels with haplotypes A, B, and C, respectively. Also included are the results of DNA sequence analyses of the G gamma and beta promoters, and of the locus-control-region hypersensitive site-2 (LCR-HS-2) of the beta-globin gene cluster in five subjects homozygous for haplotypes A, B or C; the data obtained failed to provide a satisfactory explanation for all the variations in the G gamma levels that have been observed.
Kutlar
F
F
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Huisman
T H
TH
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D005260:Female; D005319:Fetal Hemoglobin / Q000737:chemistry* / Q000235:genetics; D006239:Haplotypes; D006801:Humans; D008297:Male; D011401:Promoter Regions, Genetic; D012016:Reference Values; D013056:Spectrophotometry, Ultraviolet
D005319:Fetal Hemoglobin