title: Evaluating metal ion salts as acid hydrolase mimics: metal-assisted hydrolysis of phospholipids at lysosomal pH.
abstract: Niemann-Pick disease and drug-induced phospholipidosis are lysosomal storage disorders in which there is an excessive accumulation of sphingomyelin in cellular lysosomes. Here we have explored the possibility of developing metal-based therapeutic agents to reverse phospholipid build-up through phosphate ester bond hydrolysis at lysosomal pH (~4.8). Towards this end, we have utilized a malachite green/molybdate-based colorimetric assay to quantitate the inorganic phosphate released upon the hydrolysis of sphingomyelin by twelve d- and f-block metal ion salts. In reactions conducted at 60 °C, the yields produced by the cerium(IV) complex Ce(NH(4))(2)(NO(3))(6) were superior. An Amplex(®) Red-based colorimetric assay and mass spectrometry were then employed to detect choline. The data consistently showed that Ce(IV) hydrolyzed sphingomyelin more efficiently at lysosomal pH: i.e., yields of choline and phosphate were 54 ± 4 and 22 ± 5 % at pH ~ 4.8, compared to 8 ± 1 and 5 ± 2 % at pH ~ 7.2. Hydrolysis at 60 °C could be significantly increased by converting sphingomyelin vesicles to mixed lipid vesicles and mixed micelles of Triton X-100. We then utilized cerium(IV) to cleave sphingomyelin at 37 °C (no Triton X-100). Although choline and phosphate levels were relatively low, hydrolysis continued to be considerably more efficient at lysosomal pH. A side by side comparison to phosphatidylcholine was then made. While the yields of choline and phosphate produced by phosphatylcholine were higher, the ratio of pH ~ 4.8 hydrolysis to pH ~ 7.2 hydrolysis was usually more favorable for sphingomyelin (37 and 60 °C).
authors.affiliation: Department of Chemistry, Georgia State University, P.O. Box 4098, Atlanta, GA 30302-4098, USA.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Cepeda
authors.forename: Dominique E
authors.initials: DE
authors.lastname: Williams
authors.forename: Kathryn B
authors.initials: KB
authors.lastname: Grant
mesh_terms: D000818:Animals; D002563:Cerium / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007477:Ions / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism; D015394:Molecular Structure; D010743:Phospholipids / Q000378:metabolism*; D012492:Salts / Q000494:pharmacology; D013552:Swine
chemical_list: D007477:Ions; D010743:Phospholipids; D012492:Salts; D002563:Cerium; D006867:Hydrolases