(Received for publication, February 21, 1996, and in revised form, May 28, 1996)

From the Study Center for the Functional Biochemistry of Brain Lipids, Department of Medical Chemistry and Biochemistry, The Medical School, University of Milan, Via Saldini 50, 20133 Milano, Italy

Cultured fibroblasts from normal subjects and from subjects affected by Salla disease, characterized by the lack or misfunction of the membrane carrier responsible for the egress of sialic acid from lysosomes, were fed with ganglioside G_M3 labeled at the sialic acid acetyl group, [Neu5Ac-³H]G_M3, or at C-3 of sphingosine (Sph), [Sph-³H]G_M3, or at C-1 of stearoyl chain, [stearoyl-¹⁴C]G_M3. After a 15-h pulse the total amount of cell-bound G_M3 corresponded to about 2% of the endogenous ganglioside content. Cells were then subjected to a 72-h chase, and the radioactive products from both ganglioside catabolism and salvage processes of catabolic fragments were measured. These data indicated that about 50% of the cell-bound ganglioside underwent metabolic processing, suggesting a ganglioside half-life of 2-3 days. [Neu5Ac-³H] formed from [Neu5Ac-³H]G_M3 degradation was mostly re-cycled for the biosynthesis of gangliosides and sialoglycoproteins, only a minor part being degraded to [³H]water, which constituted only 1.6% of total metabolite linked radioactivity. [Sph-³H] from the [Sph-³H]G_M3 degradation was partly re-cycled for the biosynthesis of gangliosides, neutral glycosphingolipids and sphingomyelin, and partly (about 20% of the total metabolite linked radioactivity) degraded to [³H]water. In Salla fibroblasts metabolic processing of [Neu5Ac-³H]G_M3 produced large amounts of free [³H]Neu5Ac, and a reduced incorporation of radioactivity into glycoconjugates (as compared to normal cells). However, the accumulation of free Neu5Ac was not accompanied by an increase of tritiated water. LacCer and Cer from [stearoyl-¹⁴C]G_M3 catabolism were found to accumulate in Salla fibroblasts, an indication that the enzymes of glycosphingolipid metabolism were affected by the impairment of Neu5Ac egress from lysosomes. Particularly relevant was the accumulation of ceramide which was hardly detectable in control cells.

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