We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass (z50-100%). Analysis of LCAT kinetics parameters (V max and K m ) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [ 3 H]SM-labeled LpA-I and HDL 3 . Furthermore, expression of mutant SMase (DR608) in CHO cells revealed that DR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles. Supplementary key words high density lipoprotein . nascent LpA-I . phospholipids . sphingomyelin phosphodiesterase-1 gene . sphingomyelinase Sphingomyelin (SM) plays an important role in the structural integrity of the cellular membranes. The constitutive degradation of SM occurs in the lysosomal compartments of the cell. Fragments of the plasma membrane containing SM targeted for degradation are endocytosed and traffic through the endosomal compartments to reach the lysosomes, where they are hydrolyzed by lysosomal sphingomyelinase. The cleaved fragments (i.e., the choline head group, the fatty acids, and the sphingoid bases) then leave the lysosome and reenter the biosynthetic pathway or can be further degraded. In Niemann-Pick type I disease, which includes types A and B (NPD-A/B), lysosomal SMase is deficient, resulting in a lysosomal accumulation of SM and a secondary increase in cholesterol (1).Earlier studies by Tall and colleagues (2) investigated the crucial role of phospholipids (PLs) and their plasma transfer proteins in the metabolic pathway of HDL. It is well accepted that SM plays an important role in plasma lipoprotein metabolism. SM is the second most abundant PL in mammalian plasma. It appears in all major lipoproteins, where it is part of the monolayer of polar lipids and cholesterol that surrounds a core of neutral lipids. Up to 18% of total plasma PL occurs as SM, and the ratio of SM-to-phosphatidylcholine (PC) varies widely among lipoprotein su...

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HDLs have been proposed to have antiatherogenic properties because of their role in reverse cholesterol transport as lipid acceptors. To elucidate the phospholipid profile of these particles, we used electrospray ionization mass spectrometry to examine the phosphatidylcholine (PC) and sphingomyelin (SM) composition of HDLs purified from plasma and nascently generated in vitro from fibroblasts. We also quantitatively compared the phospholipids present in these lipoproteins between normal and Niemann-Pick disease type B (NPD-B) subjects characterized by sphingomyelinase (SMase) deficiency. We demonstrated that plasma HDLs from NPD-B were significantly enriched in SM by an average of 28%, particularly the palmitoyl SM (with an increase of 95%), which accounted for ‫ف‬ 25-44% of total SM molecular species. Similarly, we observed an increase of ‫ف‬ 63% in total SM levels in nascent HDLs prepared from NPD-B fibroblasts. Although PC levels in nascent HDLs were comparable between control and NPD-B cells, there was a 95% increase in total PC levels similar to that of SM in plasma HDLs extracted from NPD-B subjects. These data provide insight into the structure of HDLs and identify potential new roles for SMase in lipoprotein metabolism.