Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts

Scalco, F.B.; Giugliani, R.; Tobo, P.; Coelho, J.C.

doi:10.1590/S0100-879X1999000100003

Abstract

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4%, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2% DMSO (59.4 ± 9.1 and 77.0 ± 9.1 nmol h-1 mg protein-1, controls without and with 2% DMSO, respectively; 47.7 ± 5.2 and 55.8 ± 4.1 nmol h-1 mg protein-1, NPC without and with 2% DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 ± 0.049, 0.659 ± 0.041, 0.688 ± 0.063 and 0.733 ± 0.088 mg/mg protein, without DMSO, 1% DMSO, 2% DMSO and 4% DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients.

cholesterol metabolism; sphingomyelinase; Niemann-Pick disease; lysosomal storage disorders

Braz J Med Biol Res, January 1999, Volume 32(1) 23-28

Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts

F.B. Scalco², R. Giugliani¹, P. Tobo² and J.C. Coelho²

Departamentos de ¹Genética e ²Bioquímica, Universidade Federal do Rio Grande do Sul, e Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brasil

References

Abstract

Introduction

Material and Methods

Results

Discussion

Acknowledgments

Correspondence and Footnotes ^{Correspondence and Footnotes} Correspondence and Footnotes

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4%, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2% DMSO (59.4 ± 9.1 and 77.0 ± 9.1 nmol h^-1 mg protein^-1, controls without and with 2% DMSO, respectively; 47.7 ± 5.2 and 55.8 ± 4.1 nmol h^-1 mg protein^-1, NPC without and with 2% DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 ± 0.049, 0.659 ± 0.041, 0.688 ± 0.063 and 0.733 ± 0.088 mg/mg protein, without DMSO, 1% DMSO, 2% DMSO and 4% DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients.

Key words: cholesterol metabolism, sphingomyelinase, Niemann-Pick disease, lysosomal storage disorders

The Niemann-Pick group of diseases is characterized by a variable accumulation of lipids such as sphingomyelin and cholesterol. Niemann-Pick disease type C (NPC), an autosomal recessive disorder, is associated with a partial deficiency of sphingomyelinase activity and accumulation of unesterified cholesterol in the lysosomes of several tissues (1). Common clinical manifestations are neonatal jaundice, hepatomegaly, infiltration of foam macrophages in tissues, and slowly progressive neurological deterioration (2).

Despite the heterogeneity of the clinical manifestations, all NPC patients present deficient cholesterol processing in fibroblasts (2). Pentchev et al. (3) reported that excessive unesterified cholesterol is accumulated in NPC fibroblasts, suggesting a failure in the intracellular translocation of cholesterol or in its processing within lysosomal cells (4,5).

Dimethylsulfoxide (DMSO) is a dipolar organic solvent which exists in liquid form at temperatures above 18^oC. It can interact with water, salts, proteins, and lipids and can penetrate cell membranes and tissues without irreversible damage (6). DMSO has been used in clinical and biological studies and as a therapeutic agent in the treatment of some diseases (7-10).

Several investigations on the effect of DMSO on NPC fibroblast cultures have been reported. Most of these studies focused on the effect on sphingomyelinase activity (11-14). Diverging data have been reported about the effect of DMSO on the amount of intracellular cholesterol. According to Blanchette-Mackie et al. (13), incubation with 2% DMSO for 24 h does not alter the amount of intracellular cholesterol of NPC fibroblasts, whereas Yoshikawa (14) reported that 2% DMSO at the same incubation time reduces the fluorescence of unesterified cholesterol.

In the present study, NPC fibroblasts were incubated for 24 h with three different concentrations of DMSO. Our aim was to reproduce the results of other groups and to help establish whether the introduction of DMSO treatment for NPC patients is a viable option.

Cell culture

Eight normal and 7 NPC fibroblast cultures were obtained from skin biopsies. NPC cells were obtained from the Paediatric Research Unit, Guy's Hospital, London, and from patients previously diagnosed at the Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Brazil. The control cell lines were obtained from volunteers. Cells were grown in HAM-F10 medium (Gibco BRL, Gaithersburg, MD) with 10% fetal bovine serum (FBS), obtained from Gibco BRL with mycoplasma, virus and endotoxin tested, at 37^oC without 5% CO₂. When cells were confluent the flask was trypsinized and the content was transferred to cell culture dishes (NUNC) measuring 35 mm in diameter (about 20,000 cells/dish) in Eagle's minimum essential medium (MEM) supplemented with 5% (v/v) lipoprotein-deficient serum (LPDS), obtained from Gibco BRL and Sigma, respectively. These dishes were placed in a 5% CO₂ incubator at 37^oC for 4 days.

This study was approved by the Ethics Committee of Hospital de Clínicas de Porto Alegre and informed consent was obtained from all subjects that participated in this study.

DMSO addition

Following incubation, DMSO (Sigma) at one of three concentrations (1, 2 or 4%, v/v) and 50 µg/ml of culture medium (MEM) of low-density-lipoprotein (LDL, Sigma) from human plasma in 0.15 M NaCl with 0.01% (w/v) EDTA, pH 7.4, were added to each dish. Control cultures for normal and NPC fibroblasts did not contain DMSO. At this stage, cells were incubated with 5% CO₂at37^oC for 24 h.

Fluorescent cytochemical staining

Cytochemical detection of unesterified cholesterol with Filipin was carried out according to Kruth et al. (15). Slides were analyzed by fluorescence microscopy and the fibroblasts were photographed with Kodak Ektachrome film.

Lipid analysis

Cells were harvested by trypsinization and washed with saline solution. After centrifugation, the cell pellets were disrupted by freezing and thawing in liquid nitrogen. Unesterified cholesterol was measured by the method of Gamble et al. (16) and protein was measured by the method of Lowry et al. (17).

Sphingomyelinase activity

Sphingomyelinase activity was measured in order to detect the action of DMSO inside the cell. The assay was carried out with [choline-Me-¹⁴C] sphingomyelin (0.37 µCi/mmol) obtained from Du Pont (Wilmington, DE), according to the method of Pentchev et al. (18).

Statistical analysis

Data are reported as mean ± SD. The comparison among treatments using three distinct DMSO concentrations and without DMSO was performed by one-way ANOVA followed by the Duncan test whenever necessary. The Student t-test for unpaired samples was used to compare two DMSO-free groups. The analysis was performed using the statistical software package SPSS/PC+, version 3.0, and the level of significance was set at P<0.05.

NPC fibroblasts presented a large number of perinuclear granules which were not observed in normal cells (Figure 1). No effect was observed when 2% DMSO (v/v) was present in the culture medium. DMSO at 1 and 4% also did not reduce perinuclear fluorescence in NPC cells incubated for 24 h.

Sphingomyelinase activity was increased by DMSO (Table 1). Addition of 2% DMSO increased the enzyme activity in both groups, whereas 1% DMSO increased it only in normal cells. The 4% DMSO concentration reduced the cell enzyme activity significantly only in the NPC group.

Thumbnail

The results of cholesterol measurement agreed with those obtained by the Filipin staining technique. Table 2 shows that NPC cells presented a larger amount of cholesterol than normal cells. DMSO did not affect this finding at any of the concentrations used.

Thumbnail

Figure 1
- Normal and Niemann-Pick type C fibroblast cultured with LDL for 24 h with or without DMSO. Cultures were stained with Filipin for fluorescence visualization of unesterified cholesterol. A, Normal fibroblasts incubated with 50 µg/ml LDL alone; B, normal fibroblasts incubated with 50 µg/ml LDL and 2% DMSO; C, Niemann-Pick type C fibroblasts incubated with 50 µg/ml LDL alone; D, Niemann-Pick type C fibroblasts incubated with 50 µg/ml LDL and 2% DMSO. Magnification, 40X. The bar corresponds to 25 µm.

Niemann-Pick disease type C is a lipidosis characterized by a block in the intracellular translocation of exogenously derived cholesterol that differentiates it from the other types of Niemann-Pick disease, where sphingomyelinase deficiency is the primary characteristic (19).

There is no specific treatment for the disease. Some palliative treatments directed to reducing the cholesterol accumulated in the cell lysosome have been described. Sakuragawa et al. (9) and Hashimoto et al. (10) used 100-120 mg/day of DMSO for the treatment of NPC patients. Stabilization of neurologic disease was observed in an 8-year-old girl, but a 3-year-old boy showed no apparent benefit. Based on these data, no conclusion can be reached about the efficacy of treatment. Patterson et al. (20) used DMSO (100 mg/kg bid) alone or combined with cholesterol-reducing agents (lovastatin, cholestyramine and nicotinic acid) in NPC patients and concluded that DMSO alone does not reduce hepatic unesterified cholesterol in NPC patients but is effective when combined with other drugs.

Studies on the effects of DMSO on cell metabolism are very important because little is known about cholesterol homeostasis in NPC. It has been shown that DMSO corrects partial sphingomyelinase deficiency (11,12). However, regarding the reduction of cholesterol accumulated in the lysosomes of fibroblasts, results are controversial.

Blanchette-Mackie et al. (13) measured the cholesterol of NPC fibroblasts after cells were incubated with 2% DMSO and reported that the drug did not alter the amount of cholesterol when cultures were incubated for 12-24 h, but reduced the concentration of this compound after longer periods of incubation. Yoshikawa (14) observed that 2% DMSO added to the culture medium for 24 h of incubation partially reduced the amount of cholesterol in NPC cells. Up to that time, whenever the effect of DMSO on cholesterol amount was studied, 2% was the concentration utilized and the incubation time varied.

In the present study, cell cultures were incubated with DMSO for 24 h and different DMSO concentrations were used. No reduction in the amount of cholesterol was observed at any of the concentrations utilized (1, 2 and 4%). These results do not agree with those reported by Yoshikawa (14), but agree with those reported by Blanchette-Mackie et al. (13). DMSO at 2% concentration or at the other two concentrations used (1 and 4%) did not alter cholesterol inclusion in the lysosomes of NPC patients, in agreement with the unesterified cholesterol measured.

Our results confirm previous studies that have shown that 2% DMSO added to the culture medium increases sphingomyelinase activity after incubation from 6 h to 4 days (11,12). Using three different DMSO concentrations we observed that 1% DMSO is not sufficient to increase the enzyme activity in cells of NPC patients, but is sufficient to cause this effect in normal cells, and 2% DMSO increases sphyngomyelinase activity in both normal and NPC fibroblasts. We also reinforce the findings of the in vivo study of Hashimoto et al. (10) who found that DMSO, although it increases sphingomyelinase activity, fails to esterify exogenously administered cholesterol and to improve the clinical course of NPC disease.

After treatment of the cells with 4% DMSO, the enzyme activity showed a statistically significant drop in fibroblast cultures from NPC patients. In these experiments, no cell death was observed after this treatment, but 48 h of treatment with 4% DMSO caused death of more than 70% of cultured fibroblasts (data not shown), demonstrating the cytotoxic effect of 4% DMSO on cultured fibroblasts. Sakuragawa et al. (11) observed a similar effect, i.e., inhibition of cell growth, when normal cells were treated with 2% DMSO for 4 days.

If the deficiency of an enzyme involved in the degradation pathway of cholesterol is responsible for the storage of this substrate in cells of NPC patients, an increase in its activity would be expected after DMSO treatment in the same way as the effect previously shown on sphingomyelinase activity, which lowers cholesterol levels in fibroblasts. Recent studies by Carstea et al. (21) and Loftus et al. (22) showed that NPC-1 gene codes for a membrane structural protein which is involved in cholesterol transport through lysosomes and is defective in NPC patients. In this case, treatment of defective cells with DMSO would not be expected to modify intracellular cholesterol levels, as observed in our study. Even if DMSO could solubilize stored cholesterol its transport out of the lysosomes would not take place.

On the basis of present results, which are in agreement with those obtained by Blanchette-Mackie et al. (13), we conclude that DMSO probably has no effect on cholesterol levels, making no contribution to the treatment of these patients.

We wish to thank Dr. A.H. Fenson and Dr. C. Slade, Guy's Hospital, London, for sending cultured fibroblasts from NPC patients.

Address for correspondence: J.C. Coelho, Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, 90035-003 Porto Alegre, RS, Brasil. Fax: +55-51-316-8010.

Research supported by CNPq, FAPERGS and FIPE-HCPA. Received March 6, 1998. Accepted October 9, 1998.

1. Poulos A, Hudson M & Ranier E (1983). Sphingomyelinase in cultured skin fibroblasts from normal and Niemann-Pick type C patients. Clinical Genetics, 24: 225-233.
2. Pentchev PG, Comly ME, Kruth HS, Vanier MT, Wenger D, Patel S & Brady RO (1985). A defect in cholesterol esterification in Niemann-Pick disease type C patients. Proceedings of the National Academy of Sciences, USA, 82: 8247-8251.
3. Pentchev PG, Comly ME, Kruth HS, Patel S, Proestel M & Weintroub H (1986). The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease. Journal of Biological Chemistry, 261: 2772-2777.
4. Sokol J, Blanchette-Mackie EJ, Kruth HS, Dwyer NK, Amende LM, Butler JD, Robinson E, Patel S, Brady RO, Comly ME, Vanier MT & Pentchev PG (1988). Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol. Journal of Biological Chemistry, 263: 3411-3417.
5. Liscum L, Riggiero RM & Faust JR (1989). The intracellular transport of low-density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. Journal of Cell Biology, 108: 1625-1636.
6. Kharash N & Tayagarajan BS (1983). Structural basis for biological activities of dimethylsulfoxide. Annals of the New York Academy of Sciences, 411: 391-402.
7. Friend C, Scher W, Holland JG & Sato T (1971). Hemoglobin synthesis in murine virus induced leukemic cells in vitro Stimulation of erythroid differentiation by dimethylsulfoxide. Proceedings of the National Academy of Sciences, USA, 68: 378-382.
8. Kimihi Y, Palfrey C, Spector Y, Barak Y & Littauer UA (1976). Maturation of neuroblastoma cells in the presence of dimethylsulfoxide. Proceedings of the National Academy of Sciences, USA, 73: 462-466.
9. Sakuragawa N, Ohmura K, Suzuki K & Miyazato Y (1988). Clinical improvement with DMSO treatment in a patient with Niemann-Pick disease (type C). Acta Paediatrica Japonica, 30: 509-516.
10. Hashimoto K, Koeda T, Matsubara K, Ohta S, Ohno K & Ohmura K (1990). A case of type C Niemann-Pick disease. No To Hattatsu, 22: 381-384.
11. Sakuragawa N, Sato M, Yoshida Y, Kamo I, Arima M & Satoyoshi E (1985). Effects of dimethyl sulfoxide on sphingomyelinase in cultured human fibroblasts and correction of sphingomyelinase deficiency in fibroblasts from Niemann-Pick patients. Biochemical and Biophysical Research Communications, 126: 756-762.
12. Sato M, Yoshida Y, Sakuragawa N & Arima M (1988). Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts. Biochimica et Biophysica Acta, 962: 59-65.
13. Blanchette-Mackie EJ, Dwyer NK, Vanier MT, Sokol J, Merrick HF, Comly ME, Argoff CE & Pentchev PG (1989). Type C Niemann-Pick disease: dimethyl sulfoxide moderates abnormal LDL-cholesterol processing in mutant fibroblasts. Biochimica et Biophysica Acta, 1006: 219-226.
14. Yoshikawa H (1991). Effect of drugs on cholesterol esterification in normal and Niemann-Pick type C fibroblasts: AY-9944, other cationic amphiphilic drugs and DMSO. Brain and Development, 13: 115-120.
15. Kruth HS, Comly ME, Butler JD, Vanier MT, Fink JK, Wenger DA, Patel S & Pentchev PG (1986). Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous fibroblasts. Journal of Biological Chemistry, 261: 16769-16774.
16. Gamble W, Vaughan M, Kruth HS & Avigan J (1978). Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. Journal of Lipid Research, 19: 1068-1970.
17. Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193: 265-275.
18. Pentchev PG, Gal AE, Boothe AD, Omodeo-Sale F, Neumeyer JM, Quirk JM, Dawson G & Brady RO (1980). A lysosomal storage disorder in mice characterized by dual deficiency of sphingomyelinase and glucocerebrosidase. Biochimica et Biophysica Acta, 619: 669-679.
19. Pentchev PG, Vanier MT, Suzuki K & Patterson MC (1995). Niemann-Pick disease type C: a cellular cholesterol lipidosis. In: Scriver CR, Beaudet AL, Sly WS & Valle D (Editors), The Metabolic and Molecular Basis of Inherited Disease. 7th edn. McGraw-Hill, New York, 2625-2639.
20. Patterson MC, Di Bisceglie AM, Higgins JJ, Abel RB, Schiffmann R, Parker CC, Argoff CE, Grewal RP, Yu K, Pentchev PG, Brady RO & Barton NW (1993). The effect of cholesterol-lowering agents on hepatic and plasma cholesterol in Niemann-Pick disease type C. Neurology, 43: 61-64.
21. Carstea EG, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB, Nagle J, Polymeropoulos MH, Sturley SL, Ioannou YA, Higgins ME, Comly M, Cooney A, Brown A, Kaneski CR, Blanchette-Mackie EJ, Dwyer NK, Neufeld EB, Chang TY, Liscum L, Strauss III JF, Ohno K, Zeigler M, Carmi R, Sokol J, Markie D, O'Neill RR, van Diggelen OP, Elleder M, Patterson MC, Brady RO, Vanier MT, Pentchev PG & Tagle DA (1997). Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science, 277: 228-231.
22. Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA, Pentchev PG & Pavan WJ (1997). Murine model of Niemann-Pick C Disease: mutation in a cholesterol homeostasis gene. Science, 277: 232-235.

Correspondence and Footnotes

Publication Dates

Publication in this collection
06 Jan 1999
Date of issue
Jan 1999

History

Accepted
09 Oct 1998
Received
06 Mar 1998

This work is licensed under a Creative Commons Attribution 4.0 International License.

[1] 1. Poulos A, Hudson M & Ranier E (1983). Sphingomyelinase in cultured skin fibroblasts from normal and Niemann-Pick type C patients. Clinical Genetics, 24: 225-233.

[2] 2. Pentchev PG, Comly ME, Kruth HS, Vanier MT, Wenger D, Patel S & Brady RO (1985). A defect in cholesterol esterification in Niemann-Pick disease type C patients. Proceedings of the National Academy of Sciences, USA, 82: 8247-8251.

[3] 3. Pentchev PG, Comly ME, Kruth HS, Patel S, Proestel M & Weintroub H (1986). The cholesterol storage disorder of the mutant BALB/c mouse. A primary genetic lesion closely linked to defective esterification of exogenously derived cholesterol and its relationship to human type C Niemann-Pick disease. Journal of Biological Chemistry, 261: 2772-2777.

[4] 4. Sokol J, Blanchette-Mackie EJ, Kruth HS, Dwyer NK, Amende LM, Butler JD, Robinson E, Patel S, Brady RO, Comly ME, Vanier MT & Pentchev PG (1988). Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol. Journal of Biological Chemistry, 263: 3411-3417.

[5] 5. Liscum L, Riggiero RM & Faust JR (1989). The intracellular transport of low-density lipoprotein-derived cholesterol is defective in Niemann-Pick type C fibroblasts. Journal of Cell Biology, 108: 1625-1636.

[6] 6. Kharash N & Tayagarajan BS (1983). Structural basis for biological activities of dimethylsulfoxide. Annals of the New York Academy of Sciences, 411: 391-402.

[7] 7. Friend C, Scher W, Holland JG & Sato T (1971). Hemoglobin synthesis in murine virus induced leukemic cells in vitro Stimulation of erythroid differentiation by dimethylsulfoxide. Proceedings of the National Academy of Sciences, USA, 68: 378-382.

[8] 8. Kimihi Y, Palfrey C, Spector Y, Barak Y & Littauer UA (1976). Maturation of neuroblastoma cells in the presence of dimethylsulfoxide. Proceedings of the National Academy of Sciences, USA, 73: 462-466.

[9] 9. Sakuragawa N, Ohmura K, Suzuki K & Miyazato Y (1988). Clinical improvement with DMSO treatment in a patient with Niemann-Pick disease (type C). Acta Paediatrica Japonica, 30: 509-516.

[10] 10. Hashimoto K, Koeda T, Matsubara K, Ohta S, Ohno K & Ohmura K (1990). A case of type C Niemann-Pick disease. No To Hattatsu, 22: 381-384.

[11] 11. Sakuragawa N, Sato M, Yoshida Y, Kamo I, Arima M & Satoyoshi E (1985). Effects of dimethyl sulfoxide on sphingomyelinase in cultured human fibroblasts and correction of sphingomyelinase deficiency in fibroblasts from Niemann-Pick patients. Biochemical and Biophysical Research Communications, 126: 756-762.

[12] 12. Sato M, Yoshida Y, Sakuragawa N & Arima M (1988). Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts. Biochimica et Biophysica Acta, 962: 59-65.

[13] 13. Blanchette-Mackie EJ, Dwyer NK, Vanier MT, Sokol J, Merrick HF, Comly ME, Argoff CE & Pentchev PG (1989). Type C Niemann-Pick disease: dimethyl sulfoxide moderates abnormal LDL-cholesterol processing in mutant fibroblasts. Biochimica et Biophysica Acta, 1006: 219-226.

[14] 14. Yoshikawa H (1991). Effect of drugs on cholesterol esterification in normal and Niemann-Pick type C fibroblasts: AY-9944, other cationic amphiphilic drugs and DMSO. Brain and Development, 13: 115-120.

[15] 15. Kruth HS, Comly ME, Butler JD, Vanier MT, Fink JK, Wenger DA, Patel S & Pentchev PG (1986). Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous fibroblasts. Journal of Biological Chemistry, 261: 16769-16774.

[16] 16. Gamble W, Vaughan M, Kruth HS & Avigan J (1978). Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. Journal of Lipid Research, 19: 1068-1970.

[17] 17. Lowry OH, Rosebrough NJ, Farr AL & Randall RJ (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193: 265-275.

[18] 18. Pentchev PG, Gal AE, Boothe AD, Omodeo-Sale F, Neumeyer JM, Quirk JM, Dawson G & Brady RO (1980). A lysosomal storage disorder in mice characterized by dual deficiency of sphingomyelinase and glucocerebrosidase. Biochimica et Biophysica Acta, 619: 669-679.

[19] 19. Pentchev PG, Vanier MT, Suzuki K & Patterson MC (1995). Niemann-Pick disease type C: a cellular cholesterol lipidosis. In: Scriver CR, Beaudet AL, Sly WS & Valle D (Editors), The Metabolic and Molecular Basis of Inherited Disease. 7th edn. McGraw-Hill, New York, 2625-2639.

[20] 20. Patterson MC, Di Bisceglie AM, Higgins JJ, Abel RB, Schiffmann R, Parker CC, Argoff CE, Grewal RP, Yu K, Pentchev PG, Brady RO & Barton NW (1993). The effect of cholesterol-lowering agents on hepatic and plasma cholesterol in Niemann-Pick disease type C. Neurology, 43: 61-64.

[21] 21. Carstea EG, Morris JA, Coleman KG, Loftus SK, Zhang D, Cummings C, Gu J, Rosenfeld MA, Pavan WJ, Krizman DB, Nagle J, Polymeropoulos MH, Sturley SL, Ioannou YA, Higgins ME, Comly M, Cooney A, Brown A, Kaneski CR, Blanchette-Mackie EJ, Dwyer NK, Neufeld EB, Chang TY, Liscum L, Strauss III JF, Ohno K, Zeigler M, Carmi R, Sokol J, Markie D, O'Neill RR, van Diggelen OP, Elleder M, Patterson MC, Brady RO, Vanier MT, Pentchev PG & Tagle DA (1997). Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science, 277: 228-231.

[22] 22. Loftus SK, Morris JA, Carstea ED, Gu JZ, Cummings C, Brown A, Ellison J, Ohno K, Rosenfeld MA, Tagle DA, Pentchev PG & Pavan WJ (1997). Murine model of Niemann-Pick C Disease: mutation in a cholesterol homeostasis gene. Science, 277: 232-235.