Volume 57, Issue 2, Pages 446-454 (October 2000) Glycosphingolipid depletion in Fabry disease lymphoblasts with potent inhibitors of glucosylceramide synthase  Akira Abe, Lois J. Arend, Lihsueh Lee, Clifford Lingwood, Roscoe O. Brady, James A. Shayman, M.D.  Kidney International  Volume 57, Issue 2, Pages 446-454 (October 2000) DOI: 10.1046/j.1523-1755.2000.00864.x Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 1 Pathway for globotriaosylceramide metabolism. The synthesis of glucosylceramide is blocked by P4, the P4 homologues, and NBDN. α-Galactosidase A is deficient in Fabry disease resulting in globotriaosylceramide accumulation. Kidney International 2000 57, 446-454DOI: (10.1046/j.1523-1755.2000.00864.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 2 α-Galactosidase and β-hexaminidase activities in Epstein-Barr virus-transformed normal and Fabry lymphoblasts. Both enzyme activities were measured as described in the Methods section. (A and B) These show α-galactosidase and β-hexosaminidase activities, respectively. Symbols are: (▴) buffer; (•) normal; (▪) Fabry. Kidney International 2000 57, 446-454DOI: (10.1046/j.1523-1755.2000.00864.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Effects of P4 derivatives and NBDN on neutral glycosphingolipid level in Epstein-Barr virus-transformed Fabry lymphoblasts. (A) This shows a representative thin layer chromatogram of lymphoblast following inhibitor treatment for two days. The plate was developed in a two-step solvent system consisting of chloroform/methanol (98:2) as the first development and chloroform/methanol/acetic acid/water (61:33:3:3) as the second development. The lipids were visualized by charring, as described in the Methods section. Abbreviations are: CMH, ceramide monohexoside; CDH, ceramide dihexoside; CTH, ceramide trihexoside; Gb4, globoside; and SM, sphingomyelin. (B) This denotes the time-dependent changes in ceramide dihexoside (○), ceramide trihexoside (globotriaosylceramide; •), and globoside (▵) as a percentage of control glycosphingolipids. Glucosylceramide levels (CMH, □) are only shown for NBDN in which both 10 nmol/L and 10 μmol/L incubations only partially depleted the cerebroside. Kidney International 2000 57, 446-454DOI: (10.1046/j.1523-1755.2000.00864.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 3 Effects of P4 derivatives and NBDN on neutral glycosphingolipid level in Epstein-Barr virus-transformed Fabry lymphoblasts. (A) This shows a representative thin layer chromatogram of lymphoblast following inhibitor treatment for two days. The plate was developed in a two-step solvent system consisting of chloroform/methanol (98:2) as the first development and chloroform/methanol/acetic acid/water (61:33:3:3) as the second development. The lipids were visualized by charring, as described in the Methods section. Abbreviations are: CMH, ceramide monohexoside; CDH, ceramide dihexoside; CTH, ceramide trihexoside; Gb4, globoside; and SM, sphingomyelin. (B) This denotes the time-dependent changes in ceramide dihexoside (○), ceramide trihexoside (globotriaosylceramide; •), and globoside (▵) as a percentage of control glycosphingolipids. Glucosylceramide levels (CMH, □) are only shown for NBDN in which both 10 nmol/L and 10 μmol/L incubations only partially depleted the cerebroside. Kidney International 2000 57, 446-454DOI: (10.1046/j.1523-1755.2000.00864.x) Copyright © 2000 International Society of Nephrology Terms and Conditions

Figure 4 Effect of D-threo-p-OH-P4 on globotriaosylceramide expression by Fabry lymphocytes. Flow cytometric analysis of human Fabry lymphocytes treated with 50 nmol/L D-threo-p-OH-P4 (p-OH-P4) for three days. (A) Unstained control lymphocytes. Specific staining with FITC-VTB was assigned as a fluorescence intensity greater than 95% of the unstained control cells. (B) Untreated lymphocytes incubated with FITC-VTB for 40 minutes. Approximately 80% of cells were stained with VTB, which is used as a marker of globotriaosylceramide expression. Of the total stained cells, 47% are in the second peak with a log greater fluorescence intensity than the control unstained cells. (C) Lymphocytes treated for three days with p-OH-P4 and incubated with FITC-VTB. The fluorescence of the second, high-intensity peak of cells is completely eliminated by p-OH-P4, demonstrating inhibition of globotriaosylceramide expression. Kidney International 2000 57, 446-454DOI: (10.1046/j.1523-1755.2000.00864.x) Copyright © 2000 International Society of Nephrology Terms and Conditions