Sphingomyelin Derivatives Research Articles

Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy

Sphingomyelin is a key molecule of sphingolipid metabolism, and its enzymatic breakdown is associated with various infectious diseases. Here, we introduce trifunctional sphingomyelin derivatives that enable the visualization of sphingomyelin distribution and sphingomyelinase activity in infection processes. We demonstrate this by determining the activity of a bacterial sphingomyelinase on the plasma membrane of host cells using a combination of Förster resonance energy transfer and expansion microscopy. We further use our trifunctional sphingomyelin probes to visualize their metabolic state during infections with Chlamydia trachomatis and thereby show that chlamydial inclusions primarily contain the cleaved forms of the molecules. Using expansion microscopy, we observe that the proportion of metabolized molecules increases during maturation from reticulate to elementary bodies, indicating different membrane compositions between the two chlamydial developmental forms. Expansion microscopy of trifunctional sphingomyelins thus provides a powerful microscopy tool to analyze sphingomyelin metabolism in cells at nanoscale resolution.

Manipulating Redox Homeostasis of Cancer Stem Cells Overcome Chemotherapeutic Resistance through Photoactivatable Biomimetic Nanodiscs.

Tumor heterogeneity remains a significant obstacle in cancer therapy due to diverse cells with varying treatment responses. Cancer stem-like cells (CSCs) contribute significantly to intratumor heterogeneity, characterized by high tumorigenicity and chemoresistance. CSCs reside in the depth of the tumor, possessing low reactive oxygen species (ROS) levels and robust antioxidant defense systems to maintain self-renewal and stemness. A nanotherapeutic strategy is developed using tumor-penetrating peptide iRGD-modified high-density lipoprotein (HDL)-mimetic nanodiscs (IPCND) that ingeniously loaded with pyropheophorbide-a (Ppa), bis (2-hydroxyethyl) disulfide (S-S), and camptothecin (CPT) by synthesizing two amphiphilic drug-conjugated sphingomyelin derivatives. Photoactivatable Ppa can generate massive ROS which as intracellular signaling molecules effectively shut down self-renewal and trigger differentiation of the CSCs, while S-S is utilized to deplete GSH and sustainably imbalance redox homeostasis by reducing ROS clearance. Simultaneously, the depletion of GSH is accompanied by the release of CPT, which leads to subsequent cell death. This dual strategy successfully disturbed the redox equilibrium of CSCs, prompting their differentiation and boosting the ability of CPT to kill CSCs upon laser irradiation. Additionally, it demonstrated a synergistic anti-cancer effect by concurrently eliminating therapeutically resistant CSCs and bulk tumor cells, effectively suppressing tumor growth in CSC-enriched heterogeneous colon tumor mouse models.

Sphingomyelin derivatives increase the frequency of microvesicle and granule fusion in chromaffin cells

Sphingomyelin derivatives like sphingosine have been shown to enhance secretion in a variety of systems, including neuroendocrine and neuronal cells. By studying the mechanisms underlying this effect, we demonstrate here that sphingomyelin rafts co-localize strongly with synaptosomal-associated protein of 25Kda (SNAP-25) clusters in cultured bovine chromaffin cells and that they appear to be linked in a dynamic manner. In functional terms, when cultured rat chromaffin cells are treated with sphingomyelinase (SMase), producing sphingomyelin derivatives, the secretion elicited by repetitive depolarizations is enhanced. This increase was independent of cell size and it was significant 15min after initiating stimulation. Interestingly, by evaluating the membrane capacitance we found that the events in control untreated cells corresponded to two populations of microvesicles and granules, and the fusion of both these populations is clearly enhanced after treatment with SMase. Furthermore, SMase does not increase the size of chromaffin granules. Together, these results strongly suggest that SNARE-mediated exocytosis is enhanced by the generation of SMase derivatives, reflecting an increase in the frequency of fusion of both microvesicles and chromaffin granules rather than an increase in the size of these vesicles.

Toward systems epidemiology of coffee and health.

Coffee is one of the most widely consumed beverages in the world and has been associated with many health conditions. This review examines the limitations of the classic epidemiological approach to studies of coffee and health, and describes the progress in systems epidemiology of coffee and its correlated constituent, caffeine. Implications and applications of this growing body of knowledge are also discussed. Population-based metabolomic studies of coffee replicate coffee-metabolite correlations observed in clinical settings but have also identified novel metabolites of coffee response, such as specific sphingomyelin derivatives and acylcarnitines. Genome-wide analyses of self-reported coffee and caffeine intake and serum levels of caffeine support an overwhelming role for caffeine in modulating the coffee consumption behavior. Interindividual variation in the physiological exposure or response to any of the many chemicals present in coffee may alter the persistence and magnitude of their effects. It is thus imperative that future studies of coffee and health account for this variation. Systems epidemiological approaches promise to inform causality, parse the constituents of coffee responsible for health effects, and identify the subgroups most likely to benefit from increasing or decreasing coffee consumption.

A Modular Synthesis of Alkynyl-Phosphocholine Headgroups for Labeling Sphingomyelin and Phosphatidylcholine

A general route to phospho- and sphingolipids that incorporate an alkyne in the phosphocholine headgroup is described. The strategy preserves the ammonium functionality of the phosphocholine and can be easily modified to introduce desired functional groups at the N-acyl chain. The targets accessible with this strategy provide a bioorthogonal handle for postsynthetic introduction of fluorophores or other labeling agents with aqueous phase chemistry. We report the synthesis of sphingomyelin derivatives that incorporate a fluorophore and an alkyne. The modified sphingolipids retain activity as substrates for sphingomyelinase, making these compounds viable probes of enzymatic activity. Importantly, the strategy allows modification of the lipid across the phosphodiester, making the alkyne a potential probe of sphingomyelinase activity.

A lipid analogue that inhibits sphingomyelin hydrolysis and synthesis, increases ceramide, and leads to cell death

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.

Sphingosine 1-phosphate protects mouse extensor digitorum longus skeletal muscle during fatigue

Sphingomyelin derivatives exert various second messenger actions in numerous tissues. Sphingosine (SPH) and sphingosine 1-phosphate (S1P) are two major sphingomyelin derivatives present at high levels in blood. The aim of the present work was to investigate whether S1P and SPH exert relevant actions in mouse skeletal muscle contractility and fatigue. Exogenous S1P and SPH administration caused a significant reduction of tension decline during fatigue of extensor digitorum longus muscle. Final tension after the fatiguing protocol was 40% higher than in untreated muscle. Interestingly, N,N-dimethylsphingosine, an inhibitor of SPH kinase (SK), abolished the effect of supplemented SPH but not that of S1P, suggesting that SPH acts through its conversion to S1P. Moreover, SPH was not effective in Ca(2+)-free solutions, in agreement with the hypothesis that SPH action is dependent on its conversion to S1P by the Ca(2+)-requiring enzyme SK. In contrast to SPH, S1P produced its positive effects on fatigue in Ca(2+)-free conditions, indicating that S1P action does not require Ca(2+) entry and most likely is receptor mediated. The effects of S1P could be ascribed in part to its ability to prevent the reduction (-20 mV) of action potential amplitude caused by fatigue. In conclusion, these results indicate that extracellular S1P has protective effects during the development of muscle fatigue and that the extracellular conversion of SPH to S1P may represent a rheostat mechanism to protect skeletal muscle from possible cytotoxic actions of SPH.

Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells.

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.

ChemInform Abstract: Synthesis of Novel Fluorescently Labeled Sphingomyelin Derivatives Useful for Sphingomyelinase Assay.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Preparation of a naturally occurring D-erythro-(2S, 3R)-sphingosylphosphocholine using Shewanella alga NS-589

Sphingosylphosphocholine, an N-deacylated derivative of sphingomyelin, has been found to be involved in many cellular events. This paper describes a new method for preparation of a D-erythro-sphingosylphosphocholine, which is naturally occurring but difficult to prepare by chemical methods, using marine bacteria as a biocatalyst. When cultured with Shewanella alga NS-589 in synthetic medium, sphingomyelin was found to be efficiently converted by sphingomyelin deacylase to sphingosylphosphocholine. Sphingosylphosphocholine was purified with a high yield from the culture supernatant and identified to be a D-erythro-(2S,3R)-isomer containing d18:1 sphingenine as a long-chain base by fast atom bombardment mass spectrometry and NMR analyses.

Synthesis of novel fluorescently labeled sphingomyelin derivatives useful for sphingomyelinase assay

Substrates for the sphingomyelinase enzyme class have been prepared, which contain a fluorescent tag and a biotin moiety. These molecules are useful for development of rapid high throughput assays for sphingomyelinase activity. It was observed that retaining the choline ammonium group (as found in the native sphingomyelinase substrate) in synthetic substrates was critical to retain high cleavage rate by the enzyme.

Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

Toward gene therapy for Niemann-Pick disease (NPD): separation of retrovirally corrected and noncorrected NPD fibroblasts using a novel fluorescent sphingomyelin.

The neurologic (type A) and nonneurologic (type B) forms of Niemann-Pick disease (NPD) both result from deficiencies of acid sphingomyelinase (ASM) activity leading to the accumulation of sphingomyelin and other related lipids within lysosomes. Recently, the full-length cDNA and genomic sequences encoding ASM have been isolated and the nature of the molecular lesions causing NPD has been investigated. Although these developments have facilitated diagnosis for this debilitating disease, no effective treatment is currently available. Toward this latter goal, our laboratories recently reported the effectiveness of retroviral-mediated gene transfer for the in vitro correction of the cellular pathology in NPD fibroblasts (Suchi et al., 1992). In addition, novel selection procedures were developed to separate retrovirally corrected and noncorrected NPD fibroblasts based on the receptor-mediated delivery of a fluorescently (pyrene)-labeled sphingomyelin (P12-SPM) to the lysosomes of cells using liposomes coated with apolipoprotein E. In this study, we have used a different, fluorescent derivative of sphingomyelin (lissamine-rhodamine dodecanoyl sphingomyelin; LR12-SPM) to extend and improve this selection system. LR12-SPM offers a number of advantages over P12-SPM, including the facts that apolipoprotein E is not required for its efficient uptake and targeting to lysosomes and that the product of LR12-SPM degradation by ASM is efficiently transported out of cells. Thus, when analyzed in a fluorescence-activated cell sorter (FACS), there was complete separation (i.e., no overlap) of retrovirally corrected and noncorrected NPD cells after the administration of LR12-SPM.(ABSTRACT TRUNCATED AT 250 WORDS)

Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.

Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.

Sphingosine synergistically stimulates tumor necrosis factor alpha-induced prostaglandin E2 production in human fibroblasts.

Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor alpha (TNF alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF alpha signal transduction. Because TNF alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF alpha-induced PGE2 production. We found that sphingosine enhanced TNF alpha-induced PGE2 production by as much as 18-fold over TNF alpha alone. Sphingosine appeared to stimulate TNF alpha-induced PGE2 production independent of TNF alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF alpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF alpha on phospholipase A2 activity. It appears that sphingosine affects TNF alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.

Interaction of cholesterol with synthetic sphingomyelin derivatives in mixed monolayers

To study the structural requirements of the molecular interactions between cholesterol and sphingomyelins in model membranes, sphingomyelin derivatives were synthesized in which (a) the 3-hydroxy group was replaced with a hydrogen atom or with a methoxy, ethoxy, or tetrahydropyranyloxy group, (b) the N-acyl chain length was varied, and (c) the N-acyl chain length contained an alpha-hydroxy group. The chemical syntheses of these derivatives from DL-erythro-sphingosine are reported. The properties of these sphingomyelin derivatives were examined in monolayer membranes at the air/water interface. The mean molecular area of the pure N-stearoylsphingomyelin derivatives was determined, and the effects of cholesterol on the condensation of sphingomyelin packing in the monolayer were recorded. It was observed that replacement of the 3-hydroxy group of sphingomyelin with a hydrogen atom or its substitution with a methoxy or ethoxy group did not affect the ability of cholesterol to condense the molecular packing in monolayers. Even when a bulky tetrahydropyranyloxy group was introduced at the 3-hydroxy position of egg sphingomyelin, cholesterol was still able to condense the molecular packing of this derivative. The condensing effect of cholesterol on derivatives of N-stearoyl-SPMs was significantly larger than the comparable effect observed with 1,2-distearoyl-sn-glycero-3-phosphocholine or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. Our results with 3-hydroxysphingomyelins having differing N-acyl chain lengths (i.e., N-stearoyl, N-myristoyl, and N-lauroyl), and with 3-hydroxy-N-(alpha-hydroxypalmitoyl)sphingomyelin also indicated that cholesterol was able to induce condensation of the molecular packing.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of pyrenesulfonylamido-sphingomyelin and its use as substrate for determining sphingomyelinase activity and diagnosing Niemann-Pick disease

A new fluorescent derivative of sphingomyelin (PSA12-sphingomyelin) containing a pyrene-sulfonylamide residue was synthesized by covalently linking 12-((1-pyrenesulfonyl)amido)-dodecanoic acid (PSA12) to sphingosylphosphorylcholine. It was used as substrate for acidic and neutral human and murine sphingomyelinases permitting development of sensitive assays for these enzymatic activities. The product of the sphingomyelinase assay, PSA12-ceramide, could be detected in picomole quantities due to a fluorescence intensity which was 10–35-fold greater than that of other fluorescent ceramides (such as pyrene or nitrobenzoxadiazole derivatives). PSA12-sphingomyelin could be used in pure form or admixed with natural sphingomyelin; in the latter case, the enzyme hydrolyzed the fluorescent and non-fluorescent species at equal rates. Use of PSA12-sphingomyelin permitted determination of sphingomyelinase activity in cell extracts (eg human blood lymphocytes, lymphoid cell lines or cultured skin fibroblasts) as well as in hair follicles and urine. This new fluorescent derivative of sphingomyelin also permitted the detection of acid sphingomyelinase deficiency in cells derived from patients with Niemann-Pick disease.

Uptake and intracellular degradation of fluorescent sphingomyelin by fibroblasts from normal individuals and a patient with Niemann-Pick disease

Sphingomyelin, labelled with a fluorescent probe, pyrene, in the fatty acyl residue was associated with fetal calf serum; approx. 80% of the sphingomyelin was found in the low- and high-density lipoproteins. This was added to the growth medium of cultured human skin fibroblasts from normal individuals and a patient with Niemann-Pick disease type A, devoid of acid sphingomyelinase activity. The fluorescent sphingomyelin was taken up by both cell types, but only the former degraded it to produce fluorescent ceramide. Differences between normal and Niemann-Pick cells in sphingomyelin content or ceramide production were observed after several hours uptake. A more pronounced difference was noted when cells were incubated for 1 day with fluorescent sphingomyelin and then for two to three days in medium devoid of this compound. Under these conditions, the fluorescence intensity of the Niemann-Pick cells remained practically constant while that of their normal counterparts was almost completely eliminated from the cells. Comparison of fluorescence intensities of these two cell types could be made directly on aqueous suspensions of whole cells or, alternatively, on their lipid extracts. For evaluation of the degradation of fluorescent sphingomyelin to ceramide within the cells, several procedures were developed for the rapid isolation of the latter compound from the total lipid extract. The results suggest that when associated with the constituents of the fetal calf serum, sphingomyelin is taken up by the cells and transported into the lysosomal compartment where it is degraded to ceramide. Use of the fluorescent derivative of sphingomyelin provided a simple and rapid procedure for following the uptake by and degradation within the cultured cells. It also permitted the establishment of differences in the rates of degradation of the fluorescent sphingomyelin by cells with a normal metabolism and others lacking sphingomyelinase (i.e., Niemann-Pick disease type A cells).

New tools for the study of Niemann-Pick disease: analogues of natural substrate and Epstein-Barr virus-transformed lymphoid cell lines.

Acid sphingomyelinase activity was determined in Epstein-Barr virus-transformed lymphoid cell lines (LCL) established from patients affected with Niemann-Pick disease (NPD) using several substrates: sphingomyelin derivatives, radiolabeled [14C]sphingomyelin (SM), fluorescent N-(10-(1-pyrene)decanoyl)sphingomyelin (P10-SM) or colored trinitrophenylaminolauryl-sphingomyelin, and the chromogenic non-natural substrate 2-N-(hexadecanoyl)amino-4-nitrophenylphosphoryl-choline. LCL from NPD Type A and Type B showed a severe deficiency of acid sphingomyelinase determined using either substrate, whereas LCL from normal subjects had an activity close to that of blood leukocytes. Sphingomyelinase in normal LCL had the same pH optimum (5.0-5.2) and molecular form (pI 5.8) as the enzyme from other sources; identical profiles and activity levels were obtained using the various analogues of sphingomyelin. However, among these derivatives, the assay using P10-SM appeared as the most useful and sensitive for enzymatic diagnosis of NPD. Electron microscopy of NPD LCL demonstrated the lysosomal storage. These results prove the validity of LCL as an experimental model system for NPD.

A fluorometric determination of sphingomyelinase by use of fluorescent derivatives of sphingomyelin, and its application to diagnosis of Niemann-Pick disease.

Abstract We synthesized fluorescent derivatives of sphingomyelin (N-acyl-sphingosylphosphocholine) and used them as substrates for several sphingomyelinases. The following five fluorescent probes, each attached to the terminal carbon atom of the fatty acyl residue, were introduced into sphingomyelin: dansyl, pyrene, carbazole, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, and anthroic acid. We compared the rates at which the fluoro- and radiolabeled sphingomyelins were hydrolyzed. They were the same with the following three spingomyelinases: (a) a purified enzyme from Staphylococcus aureus; (b) at Triton X100-treated extract of human brain (assayed at pH 7.4 in the presence of Mg2+; and (c) aqueous extracts of brain lysosomes, skin fibroblasts, and amniotic cells, assayed at pH 5.0. Homogenates of skin fibroblasts of a patient with Niemann-Pick disease had practically no activity when assayed at pH 5, with fluorosphingomyelin as substrate. When fluorosphingomyelin was mixed in various proportions with natural sphingomyelin, enzymes from each of the three sources hydrolyzed the two substrates at equal rates. The fluorosphingomyelins can be used to estimate with great sensitivity the sphingomyelinase activity in extracts of tissues or cells, in tears, and probably in hair follicles, as well as diagnose Niemann-Pick disease, either pre- or post-natally