Evaluation of the efficiency of an assay procedure for gangliosides in human serum.
An efficiency assessment of a ganglioside assay procedure was carried out on human serum gangliosides from healthy subjects of different sex and age. The analysis of the gangliosides, extracted with chloroform/methanol and purified by lipid partitioning, ion exchange column chromatographic separation and desalting procedures as described by Senn et al. (1989) Eur J Biochem 181: 657-62, was performed by HPTLC followed by densitometric quantification. The yield of the procedure, expressed as radioactivity recovery, was determined by adding GM3 ganglioside, tritium labelled at the sialic acid acetyl group and at the C3 position of sphingosine, to the lyophilized serum or by associating it with the serum lipoproteins. In spite of the fact that the extraction and purification procedures were performed exactly as described we found the radioactivity recovery to be variable (25-50%) and much lower than that proposed. Much of the radioactivity was found in the organic phase after lipid partitioning, whilst all the ganglioside purification steps led to some further loss. After the introduction of some modifications to the procedure the recovery improved, reaching 67-79%. The analyses on 33 samples of 5 ml showed a human serum ganglioside content of about 10 nmol ml-1 (as corrected for the recovery), and confirmed that GM3 ganglioside is the main component of the total serum ganglioside mixture.
Negroni
E
E
Department of Medical Chemistry and Biochemistry, Medical School, University of Milan, Italy.
Chigorno
V
V
Tettamanti
G
G
Sonnino
S
S
D000328:Adult; D000367:Age Factors; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods; D003720:Densitometry / Q000379:methods; D005260:Female; D005679:G(M3) Ganglioside / Q000097:blood; D005732:Gangliosides / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D012016:Reference Values; D015203:Reproducibility of Results; D012727:Sex Characteristics
D005679:G(M3) Ganglioside; D005732:Gangliosides