BMP [bis(monoacylglycero)phosphate] is an acidic phospholipid and a structural isomer of PG (phosphatidylglycerol), consisting of lysophosphatidylglycerol with an additional fatty acid esterified to the glycerol head group. It is thought to be synthesized from PG in the endosomal/lysosomal compartment and is found primarily in multivesicular bodies within the same compartment. In the present study, we investigated the effect of lysosomal storage on BMP in cultured fibroblasts from patients with eight different LSDs (lysosomal storage disorders) and plasma samples from patients with one of 20 LSDs. Using ESI-MS/MS (electrospray ionization tandem MS), we were able to demonstrate either elevations or alterations in the individual species of BMP, but not of PG, in cultured fibroblasts. All affected cell lines, with the exception of Fabry disease, showed a loss of polyunsaturated BMP species relative to mono-unsaturated species, and this correlated with the literature reports of lysosomal dysfunction leading to elevations of glycosphingolipids and cholesterol in affected cells, processes thought to be critical to the pathogenesis of LSDs. Plasma samples from patients with LSDs involving storage in macrophages and/or with hepatomegaly showed an elevation in the plasma concentration of the C18:1/C18:1 species of BMP when compared with control plasmas, whereas disorders involving primarily the central nervous system pathology did not. These results suggest that the release of BMP is cell/tissue-specific and that it may be useful as a biomarker for a subset of LSDs.

INTRODUCTION

Glycerophospholipids are a broad group of biologically important lipids. They consist of a glycerol-3-phosphate backbone whose sn1 and sn2 positions are esterified with fatty acids. The phosphoryl group can be further esterified to one of several alcohol moieties including choline, ethanolamine, serine, inositol and glycerol. PG (phosphatidylglycerol; Figure 1) is an acidic phospholipid containing a glycerol moiety attached to the phosphate and is a minor component of most animal tissues; it is also an essential component of the lung surfactant [1]. BMP [bis(monoacylglycero)phosphate] is a structural isomer of PG, in which each glycerol moiety is esterified through the sn1 position to the phosphate and contains a single fatty acid ester (Figure 1). BMP is located primarily within the endosomal/lysosomal membranes of cells. It is thought to be synthesized from PG in the endosome/lysosome compartment by the action of multiple enzymes including a phospholipase A and a transacylase [2,3].

Within the lysosome, BMP is found almost exclusively in the internal vesicles, where it constitutes over 70% of phospholipids in a subpopulation of the internal membranes [4]. BMP and other anionic phospholipids act to promote the degradation of glycosphingolipids, targeted to the multivesicular bodies for degradation, by providing a suitable environment for the interaction of the glycosphingolipid hydrolases and their activator proteins with their lipid substrates [5]. BMP has also been shown to regulate cholesterol transport by acting as a collection and distribution device [6]. The acyl groups of BMP are usually enriched in polyunsaturated species [3] and this appears to be a necessary factor for the efficient partitioning of cholesterol in lipid membranes [7] and the subsequent transport of cholesterol out of the internal membranes of the multivesicular bodies [5]. Thus BMP is a critical component of the endosomal/lysosomal network and essential for the correct functioning of this system.

Structure and fragmentation of PG and BMP

Figure 1
Structure and fragmentation of PG and BMP

In positive-ion MS/MS, PG (A) undergoes a neutral loss of the ammonium adduct of the glycerophosphate moiety (189.0 Da), and BMP (B) undergoes a neutral loss of the ammonium adduct of the glycerophosphate plus the fatty acid.

Figure 1
Structure and fragmentation of PG and BMP

In positive-ion MS/MS, PG (A) undergoes a neutral loss of the ammonium adduct of the glycerophosphate moiety (189.0 Da), and BMP (B) undergoes a neutral loss of the ammonium adduct of the glycerophosphate plus the fatty acid.

The endosomal/lysosomal network is responsible for degrading macromolecules into smaller subunits for re-utilization by the cell. In a group of diseases known as LSDs (lysosomal storage disorders), such degradation is impaired and undegraded material accumulates in the lysosomes of affected cells. The primary storage material is usually the substrate for the deficient enzyme in each LSD and may consist of glycolipids such as glucosylceramide or trihexosylceramide in Gaucher disease and Fabry disease respectively, sphingomyelin or cholesterol in Niemann–Pick type A/B or C, GAGs (glycosaminoglycans) in the MPSs (mucopolysaccharidoses) or glycogen in Pompe disease. For many LSDs, the primary storage has been shown to result in impaired lysosomal function (lysosomal dysfunction) leading to the accumulation of secondary metabolites that are not substrates for the deficient enzymes [8]. In some disorders, it has been proposed that these secondarily stored materials may contribute to the pathogenesis of the disease [9,10].

In order to investigate the effect of lysosomal storage on the structure and function of the endosomal/lysosomal network, we developed a new ESI-MS/MS (electrospray ionization tandem MS)-based method for the specific determination of molecular species of BMP and PG and used this to measure these species in cultured fibroblasts and plasma from patients with a range of LSDs.

MATERIALS AND METHODS

Materials

The ISTDs (internal standards) PG C14:0/C14:0 {1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]}, PG C18:1/C18:1 {1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]}, BMP C14:0/C14:0 [sn-(3-myristoyl-2-hydroxy)-glycerol-1-phospho-sn-3′-(1′-myristoyl-2′-hydroxy)-glycerol] and BMP C18:1/C18:1 [sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3′-(1′-oleoyl-2′-hydroxy)-glycerol] were obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.). All solvents were of HPLC grade and were used without further purification.

Patient samples

The Children, Youth and Women's Health Service, Research Ethics Committee (Adelaide, SA, Australia) approved the use of skin fibroblasts and plasma in these studies. Control plasma and cultured skin fibroblasts were collected from patients, with informed consent, and were sent to the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Diseases (Department of Genetic Medicine, Women's and Children's Hospital, Adelaide, Australia) for testing. Samples used in the present study were de-identified to the researchers.

Cell culture

Human diploid fibroblasts were established from skin biopsies submitted to the Department of Genetic Medicine for diagnosis [11]. Skin fibroblasts from unaffected individuals and LSD patients were maintained in BME (basal medium Eagle) supplemented with 10% (v/v) foetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. Cells were harvested at 2 weeks post-confluence and cell extracts were prepared in 200 μl of 20 mmol/l Tris/HCl and 0.5 mol/l NaCl (pH 7.2) by sonication for 20 s. Total cell protein was determined by the method of Lowry et al. [12].

Plasma fractionation

Plasma was fractionated into VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein), HDL (high-density lipoprotein) and lipoprotein-depleted plasma by differential ultracentrifugation using potassium bromide [13]. Plasma was diluted with an equal volume of PBS and then centrifuged (10000 g and 30 min) to remove cellular debris. Exosomes were then isolated by ultracentrifugation (110000 g and 70 min) [14].

Extraction of phospholipids

Extraction of PG and BMP from fibroblast extracts and plasma was performed by the method of Folch et al. [15]. Briefly, fibroblast extracts (100 μl) or plasma (100 μl) were extracted with 2.0 ml of chloroform/methanol (2:1, v/v) containing 400 pmol of each ISTD (PG C14:0/C14:0 and BMP C14:0/C14:0). The mixture was shaken for 10 min and then allowed to stand at room temperature (20 °C) for a further 50 min. The samples were partitioned with the addition of 0.4 ml of water and shaken for 10 min. To facilitate phase separation, the mixture was centrifuged (2500 g for 5 min). The lower hydrophobic layer containing the phospholipids was transferred to a clean glass test tube and dried under a gentle stream of nitrogen. This phospholipid extract was reconstituted in 100 μl of 10 mmol/l ammonium formate in methanol.

MS analysis of PG and BMP

Phospholipid analysis was performed using a PE Sciex API 3000 triple-quadrupole mass spectrometer with a turbo-ionspray source (200 °C), a Gilson 233 autosampler and the Analyst 1.1 data acquisition system. N2 was used as the collision gas at a pressure of 2×10−5 torr (1 torr=0.133 kPa).

In order to identify the molecular species of PG and BMP in cultured skin fibroblasts, flow-injection experiments were performed in negative-ion mode. Samples (20 μl) were injected at a flow rate of 80 μl/min using 10 mmol/l ammonium formate in methanol as the mobile phase. Precursor-ion scans for the mass to charge ratio (m/z) 153.0 corresponding to the glycerophosphate moiety and for the m/z corresponding to individual fatty acid moieties [i.e. m/z 281.3 for oleic acid (C18:1)] identified individual PG/BMP species.

Quantification of individual PG and BMP species was performed in positive-ion mode by LC (liquid chromatography)– ESI-MS/MS on an HP 1100 HPLC system (Hewlett–Packard). Samples (20 μl) were injected on to an Alltima C18, 3 μm, 50 mm×2.1 mm column and were eluted with an isocratic flow (200 μl/min) of 10 mmol/l ammonium formate in methanol. PG species were quantified by measuring the neutral loss of 189 Da, corresponding to the ammonium adduct of the glycerophosphate (Figure 1). Similarly, BMP species were quantified by measuring the neutral loss corresponding to the monoacylglycerophosphate moiety from each parent molecular species (Figure 1). Multiple PG and BMP species, containing different acyl chains, were measured in each experiment by using MRM (multiple-reaction monitoring); each ion pair was monitored for 50 ms with a resolution of 0.7 a.m.u. (atomic mass units) at half-peak height. PG and BMP concentrations were calculated by relating the peak areas of each species to the peak area of the corresponding ISTD by using Analyst 1.4.1 quantification software. PG species were related to PG C14:0/C14:0 and the BMP species were related to BMP C14:0/C14:0.

RESULTS

CID (collision-induced dissociation) of PG and BMP

CID of PG C14:0/C14:0 and BMP C14:0/C14:0 in negative-ion mode produced similar fragmentation patterns for both compounds (Figures 2A and 2C), making specific determination of either lipid difficult in the presence of the other. Intense product ions were observed for the myristate (C14:0) fatty acid moieties (m/z 227.1) and for the glycerophosphate fragment at m/z 153.0. Minor differences were observed for the product ions at m/z 437.4 and 455.4 corresponding to the neutral loss of the acyl chain ± water. One clear distinction between PG and BMP was the ability of PG to lose the glycerol moiety without the loss of a fatty acid. Thus the neutral loss of 74.0 Da is specific for the PG species only, although this represented only a minor fragmentation product and was not suitable for specific quantification of the PG species.

Product-ion spectra for PG and BMP

Figure 2
Product-ion spectra for PG and BMP

CID of PG C14:0/C14:0 in negative-ion mode (A) and positive-ion mode (B) was performed with collision energy set to –55 and 25 V respectively. CID of BMP C14:0/C14:0 in negative-ion mode (C) and positive-ion mode (D) was performed by using the same conditions.

Figure 2
Product-ion spectra for PG and BMP

CID of PG C14:0/C14:0 in negative-ion mode (A) and positive-ion mode (B) was performed with collision energy set to –55 and 25 V respectively. CID of BMP C14:0/C14:0 in negative-ion mode (C) and positive-ion mode (D) was performed by using the same conditions.

In positive-ion mode, the CID patterns for PG and BMP were different (Figures 2B and 2D). The positive-ion ammonium adducts of PG C14:0/C14:0 and BMP C14:0/C14:0 both have precursor ion signals at m/z 684.5, but only the PG species can undergo a neutral loss of 189.0 Da, corresponding to the ammonium adduct of the glycerophosphate moiety, resulting in the diacylglycerol fragment (m/z 495.5). In contrast, although it is possible for both PG and BMP to produce a monoacylglycerol fragment (m/z 285.3), this fragmentation occurs more readily in the BMP species (Figure 2D). PG species containing different acyl chains gave similar responses for the neutral loss of 189.0 Da, as determined by the relative responses for PG C14:0/C14:0 and PG C18:1/C18:1 standards. BMP species also gave similar responses for the loss of the monoacylglycerol phosphate moiety, as determined by comparison of the signals from the BMP C14:0/C14:0 and BMP C18:1/C18:1 standards. However, unlike the PG species, fragmentation of BMP to produce the monoacylglycerol ion can occur on either side of the phosphate, leading to two different product ions for asymmetrical BMP species. This reduced the signal from the asymmetric BMP species compared with the symmetrical species. This was confirmed by comparison of the Q1 signals and the precursor-ion signals for a fibroblast extract, where the symmetrical BMP species resulted in product-ion signals that were approx. 24% of the Q1 signal, while asymmetrical species gave product-ion signals that were approx. 12% of the Q1 signal. To correct for this difference, signals resulting from asymmetric BMP species were multiplied by a factor of two, prior to the calculation of BMP concentrations.

Identification of PG and BMP species in cultured skin fibroblasts

Precursor-ion scanning in negative-ion mode for the common glycerophosphate/water fragment (m/z 153.0) and for the m/z corresponding to the fatty acid chains allowed us to identify the most abundant PG/BMP species in skin fibroblasts. This was done in the manner described by Han et al. [16] using a two-dimensional ESI-MS/MS approach to identify cross-peak intensities that correspond to the phospholipid species observed in the precursor of m/z 153.0 spectra. As an example, precursor scanning for m/z 153.0 identified a signal at m/z 775.5 that could result from a PG or BMP C36:1 species. Confirmation of the presence of the C18:0/C18:1 species was made by conducting precursor-ion scans for the fatty acid products C18:0 (m/z 283.3) and C18:1 (m/z 281.3) respectively and identifying the parent ion at m/z 775.5. Fatty acid species identified on the PG/BMP species included C16:0, C16:1, C18:0, C18:1, C18:2, C20:3, C20:4, C22:4, C22:5 and C22:6. While most combinations of fatty acids could be identified, many were minor components. No distinction was made between PG and BMP species in these analyses.

The PG/BMP species identified in negative-ion mode were used to define MRM experiments for both PG and BMP species in positive-ion mode. Subsequent analyses of skin fibroblast extracts in positive-ion mode identified the major PG species in control skin fibroblasts as PG C36:1, which represented species containing primarily C18:0 and C18:1 fatty acids, and PG C34:1, which represented a mixture of PG species containing either C16:1 and C18:0 or C16:0 and C18:1 fatty acids. Other minor species include PG C34:2, PG C36:2, PG C36:3, PG C38:5 and PG C40:5. PG species with two long-chain polyunsaturated fatty acids were present in relatively minor amounts. In contrast, the prominent BMP species of control fibroblasts were BMP C18:1/C22:6, BMP C22:5/C22:6 and BMP C22:6/C22:6. BMP C18:0/C18:1 constituted less than 1% of the total BMP in the control fibroblast extracts.

Quantification of PG and BMP by LC ESI-MS/MS

Analysis of PG and BMP, in cell or plasma extracts, by flow injection in positive-ion mode, resulted in greater than 98% signal suppression. To overcome this suppression effect, an LC step was incorporated into the analysis to separate the PG and BMP from other more easily ionized lipids and salts; this reduced the suppression to approx. 50% for both PG and BMP and resulted in a limit of detection in plasma of 2 and 6 nmol/l for PG and BMP respectively. The most likely source of the signal suppression was thought to be the abundant PC (phosphatidylcholine) species; monitoring of the PC species by precursor ion scanning of m/z 184 demonstrated that the major species (PC C32:1, C34:1, C34:2 and C36:2) were eluted after the PG and BMP species and so had no influence on signal suppression when the LC step was used (results not shown). Retention times of the PG and BMP species on the LC column were in the range of 1.7–5.5 min. Figure 3 shows chromatograms of PG C36:2 and BMP C18:1/C18:1 species in control and Gaucher plasma extracts with PG C14:0/C14:0 and BMP C14:0/C14:0 ISTDs. The double peaks observed for both BMP C14:0/C14:0 and BMP C18:1/C18:1 (Figure 3B) were thought to result from structural isomers of BMP with different acylation of the glycerol moieties. The PG C14:0/C14:0 ISTD shows a single peak since only one structural isomer can exist. However, the PG C36:2 MRM chromatogram shows multiple peaks, which represent several PG species, which could include PG C18:1/C18:1, PG C18:0/C18:2 and PG C16:0/C20:2.

Analysis of PG C36:2 and BMP C18:1/C18:1 by LC-MS/MS

Figure 3
Analysis of PG C36:2 and BMP C18:1/C18:1 by LC-MS/MS

Lipid extracts of plasma samples from a control individual and a Gaucher patient were analysed as described in the Materials and methods section. The signals corresponding to PG C36:2 (MRM=m/z 792.5/603.5) were normalized to the signal from the ISTD PG C14:0/C14:0 (MRM=m/z 684.5/495.5) (A). The signals corresponding to BMP C18:1/C18:1 (MRM=m/z 792.5/339.4) from each sample were normalized to the signal from the ISTD BMP C14:0/C14:0 (MRM=m/z 684.5/285.3) (B).

Figure 3
Analysis of PG C36:2 and BMP C18:1/C18:1 by LC-MS/MS

Lipid extracts of plasma samples from a control individual and a Gaucher patient were analysed as described in the Materials and methods section. The signals corresponding to PG C36:2 (MRM=m/z 792.5/603.5) were normalized to the signal from the ISTD PG C14:0/C14:0 (MRM=m/z 684.5/495.5) (A). The signals corresponding to BMP C18:1/C18:1 (MRM=m/z 792.5/339.4) from each sample were normalized to the signal from the ISTD BMP C14:0/C14:0 (MRM=m/z 684.5/285.3) (B).

The neutral loss of the ammonium adduct of the glycerophosphate moiety, which was used to monitor the PG species in positive-ion mode, cannot occur in the BMP species as both glycerol structures are acylated. However, analysis of the BMP C14:0/C14:0 ISTD did show a small signal corresponding to the neutral loss of a glycerophosphate moiety. The elution time of this signal (2.1 min) coincided with the BMP C14:0/C14:0 ISTD and so precluded the possibility of a contamination of the BMP ISTD with PG C14:0/C14:0, which had an elution time of 1.9 min. This signal represented approx. 2% of the BMP signal and is thought to result from in-source rearrangement of an acyl group to form PG C14:0/C14:0 (results not shown). We also observed a small signal from the PG C14:0/C14:0 ISTD, corresponding to the monoacylglycerol product determined by the MRM (m/z 685/285). The collision energy used for the quantification of BMP species was subsequently optimized to minimize the intensity of the signal from the PG species while maintaining the signal intensity for the BMP species. The resulting signal from the PG species using the BMP-specific MRM was approx. 2% of the signal observed in the PG-specific MRM (neutral loss of 189.0 Da; results not shown). The response of BMP in the PG-specific MRM and the PG in the BMP-specific MRM did not affect quantification for most species as the chromatographic step provided adequate separation of the corresponding PG and BMP species. However, LC analysis of PG C40:8, PG C42:10, PG C44:10, PG C44:11 and PG C44:12 did not provide complete separation from the corresponding BMP species as they all eluted between 1.7 and 2.0 min. Consequently, for quantification, each of these PG species was corrected for the contribution of the corresponding BMP species and each BMP species was corrected for the contribution of the corresponding PG species.

Assay performance

The PG/BMP assay was linear within the range 2–2000 pmol/ml (R2>0.999). Intra- and inter-assay CVs (coefficients of variation) were determined for each analyte using 100 μg of cell lysate per assay. Intra-assay CVs were calculated from five repeats of the same sample and inter-assay CVs were calculated from 30 repeats performed over 6 days. Intra-assay CVs were less than 10% and inter-assay CVs less than 15% for all PG and BMP species present at concentrations greater than 2.0 pmol/100 μg of cell protein (30 of 36 species).

PG and BMP amounts in cultured skin fibroblasts

Analysis of fibroblast extracts from LSD patients showed relatively minor changes in the amount of PG (Table 1). In addition, the relative amounts of each PG species (PG profile) in most LSD cell lines were almost identical with the control fibroblasts (compare Figures 4A and 4G), the exception being Pompe fibroblasts (Figure 4E), which had a decreased amount of PG C36:1 compared with control cells and corresponding increased concentrations of PG C34:1 and PG C36:2. LSD fibroblasts also showed only a modest increase (2–3-fold) in total BMP amounts compared with control fibroblasts (Table 1), although this was greater in Fabry cell lines (5–9-fold). However, in all cell lines, with the exception of the MPS IIIA cell line, there was a substantial increase in the amount of BMP C18:1/C18:1 (3–20-fold). In contrast with the BMP C18:1/C18:1 species, most cell lines showed no increase in the major BMP species, BMP C22:6/C22:6, with several cell lines showing a decrease in this species (Table 1). Thus, with the exception of Fabry disease, all LSD cell lines showed a different BMP profile compared with the control cell lines (compare Figures 4B with 4H); in particular, Niemann–Pick type C cell lines had almost no BMP species containing two polyunsaturated fatty acids (Figure 4F).

Table 1
PG and BMP in control and LSD skin fibroblasts

Values are means (S.D.) and are expressed as pmol/mg of cell protein. *Significantly different from the control (P<0.05, Student's t test, equal variance not assumed).

Disorder Total PG Total BMP BMP 18:1/18:1 BMP 22:6/22:6 
Control (n=7) 135 (41) 602 (110) 20 (6) 146 (34) 
Fabry (n=3) 211 (32)* 4392 (1159)* 231 (75)* 898 (224)* 
Gaucher (n=2) 299 (10) 430 (172) 85 (42) 35 (11)* 
MPS I (n=3) 325 (69) 1430 (241) 310 (101)* 103 (18)* 
MPS II (n=2) 424 (87) 1426 (306) 326 (85) 99 (8)* 
MPS IIIA (n=1) 96 84 18 
Niemann–Pick type A/B (n=3) 219 (75) 1514 (1018) 249 (122) 133 (100) 
Niemann–Pick type C (n=2) 88 (23) 342 (51) 95 (10)* 2 (3)* 
Pompe (n=1) 595 1006 215 43 
Disorder Total PG Total BMP BMP 18:1/18:1 BMP 22:6/22:6 
Control (n=7) 135 (41) 602 (110) 20 (6) 146 (34) 
Fabry (n=3) 211 (32)* 4392 (1159)* 231 (75)* 898 (224)* 
Gaucher (n=2) 299 (10) 430 (172) 85 (42) 35 (11)* 
MPS I (n=3) 325 (69) 1430 (241) 310 (101)* 103 (18)* 
MPS II (n=2) 424 (87) 1426 (306) 326 (85) 99 (8)* 
MPS IIIA (n=1) 96 84 18 
Niemann–Pick type A/B (n=3) 219 (75) 1514 (1018) 249 (122) 133 (100) 
Niemann–Pick type C (n=2) 88 (23) 342 (51) 95 (10)* 2 (3)* 
Pompe (n=1) 595 1006 215 43 

PG and BMP profiles from control and LSD skin fibroblasts

Figure 4
PG and BMP profiles from control and LSD skin fibroblasts

Control skin fibroblasts (n=7) and LSD skin fibroblasts (n=17) were analysed for PG and BMP species by LC ESI-MS/MS as described in the Materials and methods section. The relative amount of each species of PG and BMP is shown as a percentage of the total with error bars indicating one S.D. Control PG and BMP are shown in (A) and (B) respectively. PG and BMP species present in Fabry skin fibroblasts are shown in (C) and (D) respectively. PG species in Pompe skin fibroblasts are shown in (E) and BMP species in Niemann–Pick type C skin fibroblasts in (F). PG species present in the remaining LSD skin fibroblasts [Gaucher (n=2), MPS I (n=3), MPS II (n=2), MPS IIIA (n=1), Niemann–Pick type A/B (n=3) and Niemann–Pick type C (n=2)] are shown in (G) and BMP in the remaining skin fibroblasts [Gaucher (n=2), MPS I (n=3), MPS II (n=2), MPS IIIA (n=1), Niemann–Pick type A/B (n=3) and Pompe (n=1)] are shown in (G).

Figure 4
PG and BMP profiles from control and LSD skin fibroblasts

Control skin fibroblasts (n=7) and LSD skin fibroblasts (n=17) were analysed for PG and BMP species by LC ESI-MS/MS as described in the Materials and methods section. The relative amount of each species of PG and BMP is shown as a percentage of the total with error bars indicating one S.D. Control PG and BMP are shown in (A) and (B) respectively. PG and BMP species present in Fabry skin fibroblasts are shown in (C) and (D) respectively. PG species in Pompe skin fibroblasts are shown in (E) and BMP species in Niemann–Pick type C skin fibroblasts in (F). PG species present in the remaining LSD skin fibroblasts [Gaucher (n=2), MPS I (n=3), MPS II (n=2), MPS IIIA (n=1), Niemann–Pick type A/B (n=3) and Niemann–Pick type C (n=2)] are shown in (G) and BMP in the remaining skin fibroblasts [Gaucher (n=2), MPS I (n=3), MPS II (n=2), MPS IIIA (n=1), Niemann–Pick type A/B (n=3) and Pompe (n=1)] are shown in (G).

PG and BMP concentrations in plasma

Plasma samples from 20 control individuals and 64 LSD-affected individuals representing 20 LSDs were analysed for ten PG species and ten BMP species by the LC–ESI-MS/MS method. The profile of PG in plasma was different from that observed in fibroblasts. In control plasma, the most abundant species were PG C34:1, PG C36:1 and PG C36:2 (Figure 5A). The BMP profile from the control individuals was also substantially different from that observed in the cultured cells (Table 2; Figure 5B). The major species in plasma was BMP C18:1/C18:1, with significant amounts of the shorter-chain species BMP C16:0/C16:0 and BMP C16:0/C18:1, while the major long-chain species observed in fibroblasts (BMP C22:6/C22:6) was present in only minor amounts. Only the Niemann–Pick type A patients showed a significant increase in the concentration of plasma PG as determined by the Mann–Whitney U test (Table 2). In contrast, five of the 20 LSDs showed a significant increase in the concentration of plasma BMP, and examination of the BMP profile for Niemann–Pick type A/B showed that this was primarily an increase in the concentrations if BMP C18:1/C18:1 and BMP C18:1/C18:2 (Figure 5D). When the Mann–Whitney U test was applied to the BMP C18:1/C18:1 data, ten LSDs show a significant increase (P<0.05) compared with the control group (Table 2).

PG and BMP profiles for control and Niemann–Pick type A/B plasma samples

Figure 5
PG and BMP profiles for control and Niemann–Pick type A/B plasma samples

Plasma samples from control individuals (n=20; A, B) and Niemann–Pick type A/B patients (n=6; C, D) were analysed for PG and BMP species by LC ESI-MS/MS as described in the Materials and methods section. The amount of each species of PG and BMP is shown as a percentage of the total with the error bars indicating one S.D.

Figure 5
PG and BMP profiles for control and Niemann–Pick type A/B plasma samples

Plasma samples from control individuals (n=20; A, B) and Niemann–Pick type A/B patients (n=6; C, D) were analysed for PG and BMP species by LC ESI-MS/MS as described in the Materials and methods section. The amount of each species of PG and BMP is shown as a percentage of the total with the error bars indicating one S.D.

Table 2
PG and BMP concentrations in control and LSD plasma

Values are means (S.D.) and are expressed as nM. GM1, GM1 gangliosidosis; MLD, metachromatic leukodystrophy; ML II/III, mucolipidosis type II/III; JNCL, juvenile neuronal ceroid lipofuscinosis; LINCL, late infantile neuronal ceroid lipofuscinosis; MSD, multiple sulfatase deficiency; M–W U, value from a Mann–Whitney U test for each group compared with the control group. *P<0.05.

Patient group n Total PG M–W U Total BMP M–W U BMP 18:1/18:1 M–W U 
Control 20 456 (278) 197 (80) 47 (12)    
Fabry 316 (110) 50 167 (60) 61 35 (11) 40* 
Gaucher 415 (166) 59 1297 (615) 0* 700 (362) 0* 
GM1 194 (14) 4* 305 (85) 8* 130 (60) 1* 
Krabbe 218 (111) 12 166 (123) 29 25 (13) 6* 
MLD 205 (66) 8* 128 (10) 38 (10) 17 
MLII/III 218 (134) 8* 222 (43) 21 40 (18) 22 
MPS I 297 (138) 33 270 (104) 24 125 (86) 4* 
MPS II 206 (127) 8* 290 (72) 108 (63) 5* 
MPS IIIA 253 (156) 11 198 (39) 26 52 (21) 30 
MPS IV 293 (175) 21 202 (68) 25 52 (19) 22 
MPS VI 423 10 70 13 
MPS VII 191 228 104 0* 
N–P A 1087 (369) 2* 10516 (4606) 0* 6055 (3274) 0* 
N–P B 529 (378) 35 4165 (3605) 0* 1920 (1963) 0* 
N–P C 389 (181) 36 531 (77) 0* 264 (51) 0* 
Sandhoff 408 (280) 28 240 (52) 16 81 (10) 1* 
Tay–Sachs 320 (79) 19 204 (31) 21 66 (16) 7* 
JNCL 211 (65) 81 (18) 0* 10 (1) 0* 
LINCL 249 (34) 13 174 (21) 27 40 (12) 19 
MSD 661 271 79 
Patient group n Total PG M–W U Total BMP M–W U BMP 18:1/18:1 M–W U 
Control 20 456 (278) 197 (80) 47 (12)    
Fabry 316 (110) 50 167 (60) 61 35 (11) 40* 
Gaucher 415 (166) 59 1297 (615) 0* 700 (362) 0* 
GM1 194 (14) 4* 305 (85) 8* 130 (60) 1* 
Krabbe 218 (111) 12 166 (123) 29 25 (13) 6* 
MLD 205 (66) 8* 128 (10) 38 (10) 17 
MLII/III 218 (134) 8* 222 (43) 21 40 (18) 22 
MPS I 297 (138) 33 270 (104) 24 125 (86) 4* 
MPS II 206 (127) 8* 290 (72) 108 (63) 5* 
MPS IIIA 253 (156) 11 198 (39) 26 52 (21) 30 
MPS IV 293 (175) 21 202 (68) 25 52 (19) 22 
MPS VI 423 10 70 13 
MPS VII 191 228 104 0* 
N–P A 1087 (369) 2* 10516 (4606) 0* 6055 (3274) 0* 
N–P B 529 (378) 35 4165 (3605) 0* 1920 (1963) 0* 
N–P C 389 (181) 36 531 (77) 0* 264 (51) 0* 
Sandhoff 408 (280) 28 240 (52) 16 81 (10) 1* 
Tay–Sachs 320 (79) 19 204 (31) 21 66 (16) 7* 
JNCL 211 (65) 81 (18) 0* 10 (1) 0* 
LINCL 249 (34) 13 174 (21) 27 40 (12) 19 
MSD 661 271 79 

BMP localization in plasma

Fractionation of plasma samples from control individuals (n=3) and Gaucher patients (n=3) into VLDL, LDL, HDL and lipoprotein-deficient plasma followed by quantification of BMP C18:1/C18:1 in each fraction showed that, in control plasma, BMP was present in all plasma compartments with approx. 40% associated with lipoproteins (10% in VLDL, 22% in LDL and 9% in HDL) and 60% remaining in the lipoprotein-deficient plasma. In contrast, plasma from Gaucher patients had approx. 80% of BMP associated with the lipoprotein fraction (55% in VLDL, 15% in LDL and 10% in HDL) and only 20% remaining in the lipoprotein-deficient plasma. No detectable BMP could be isolated in exosomes from either control individuals or Gaucher patients.

DISCUSSION

To investigate the effect of lysosomal storage on BMP, we sought to develop a rapid sensitive method for the specific quantification of individual BMP species in complex mixtures that also contain its structural isomer, PG. Analysis of molecular species of BMP has previously been performed by fast-atom bombardment MS after separation of the PG and BMP species by TLC [17]. More recently, Kakela et al. [18] used LC–ESI-MS in negative-ion mode to quantify molecular species of BMP. This method relied on the separation of the PG and BMP by LC as they were not differentiated by MS in negative-ion mode; consequently, each analysis required greater than 30 min to complete. In an analysis of lipid remodelling in essential fatty acid-deficient mice, Duffin et al. [19] used the neutral loss of 189.0 Da in positive-ion mode to identify the ammonium adducts of PG species. In the present study, we performed ESI-MS/MS analysis of both PG and BMP species in positive-ion mode, which resulted in different CID patterns and enabled quantification of both PG and BMP species without the need for prior separation. We combined this with a short (10 min) LC step, to reduce signal suppression and improve the sensitivity of the quantification.

The major species of BMP in cultured skin fibroblasts contained primarily mono-unsaturated (C18:1) and polyunsaturated (C22:5 and C22:6) fatty acids with BMP C22:6/C22:6 being the most abundant. These species were also identified by Kakela et al. [18] in brain tissue of patients with infantile and juvenile neuronal ceroid lipofuscinoses, both LSDs. This fatty acid composition is unusual in that while other phospholipids contain polyunsaturated species it is usually in combination with a saturated fatty acid [18]. In contrast with BMP, the major PG species contained primarily mono-unsaturated (C18:1 and C16:1) and saturated (C18:0 and C16:0) fatty acids. We observed relatively small changes in the total BMP amounts within LSD-affected cells, although in addition to BMP increases in Niemann–Pick type A/B cells, reported previously [20], we also observed increases in MPS I, MPS II and Fabry disease cells. More importantly, all cell lines, with the exception of Fabry disease, showed an altered BMP profile regardless of the degree of BMP elevation. The change in the BMP profile was consistent for most LSD cell lines (Figure 4H) but was most striking in the Niemann–Pick type C cell lines, which had the greatest reduction in the amount of polyunsaturated BMP species (Figure 4F). Altered lysosomal function associated with these changes in BMP composition as is evident from the reports of both glycosphingolipid and cholesterol accumulation in a range of LSD types, which include not only lipid storage disorders but also disorders such as the MPS where the primary storage is GAG [8,21]. The observed reduction in the amount of polyunsaturated BMP species is likely to alter the availability of glycolipids for interaction with saposins and the glycosidases required for their degradation [5,22] and will affect cholesterol partitioning within these membranes [7] thereby reducing their ability to remove cholesterol from the lysosome. These changes in BMP composition resulting from lysosomal storage may therefore provide the mechanistic link between the primary storage and the secondary accumulation of lipids and cholesterol, which appears to be a critical event in the pathogenesis of many LSDs.

There are a number of possible mechanisms whereby lysosomal storage may induce changes in the BMP profile. Lysosomal storage may result in alterations in lipid and protein trafficking within the endosomal/lysosomal compartment, thereby influencing the availability of either enzymes or substrates involved in BMP synthesis. Alternatively, alterations in endosomal/lysosomal pH resulting from lysosomal storage may affect the specificity of the enzymes involved in BMP synthesis. In any event, this would appear to be a general result of endosomal/lysosomal storage and not dependent on the material stored. The exception to this observation is Fabry disease where the accumulation of trihexosylceramide resulted in the largest increase in total BMP (5–7-fold) but did not affect the BMP profile (Figure 4D). Interestingly, while many LSDs, including Gaucher disease, Niemann–Pick disease types A and C and the MPSs, result in secondary accumulation of gangliosides in neurons, neuronal storage in Fabry disease appears limited and does not involve alterations in ganglioside expression [8].

In light of the ubiquitous nature of the alterations of BMP in LSD, we quantified PG and BMP in plasma from control and LSD-affected individuals to further investigate the nature of the changes in BMP associated with LSD. The differences in the plasma BMP species compared with the fibroblasts suggests that secretion of BMP into the circulation may be fatty acid-dependent with a preference for saturated and mono-unsaturated fatty acids. The LSDs resulting in the highest elevation of plasma BMP C18:1/C18:1 include Gaucher disease, Niemann–Pick type A/B and Niemann–Pick type C; this suggests that, at least in part, BMP is coming from macrophages, which are the primary site of storage in these disorders. Other disorders, including MPS I and MPS II, also showed a significant elevation of BMP C18:1/C18:1, suggesting that other cell types And tissues may also be contributing to the plasma BMP, in these disorders. Fractionation of plasma demonstrated that approx. 40% of BMP C18:1/C18:1 was associated with lipoprotein particles in control plasma and that this was greater (80%) in Gaucher disease. This suggests that the transport of BMP out of liver cells in the form of newly synthesized VLDL is likely to be a major source of plasma BMP. In addition, the presence of BMP in HDL indicates that release of BMP from macrophages, which are known to release cholesterol and phospholipids into HDL via a number of mechanisms [2325], may also contribute to plasma BMP.

This work was supported by the Women's and Children's Hospital Research Foundation (Australia), the National Health and Medical Research Council (Australia) and Genzyme.

Abbreviations

     
  • BMP

    bis(monoacylglycero)phosphate

  •  
  • BMP

    C14:0/C14:0, sn-(3-myristoyl-2-hydroxy)-glycerol-1-phospho-sn-3′-(1′-myristoyl-2′-hydroxy)-glycerol

  •  
  • BMP

    C18:1/C18:1, sn-(3-oleoyl-2-hydroxy)-glycerol-1-phospho-sn-3′-(1′-oleoyl-2′-hydroxy)-glycerol

  •  
  • CID

    collision-induced dissociation

  •  
  • CV

    coefficient of variation

  •  
  • ESI-MS/MS

    electrospray ionization tandem MS

  •  
  • GAG

    glycosaminoglycan

  •  
  • HDL

    high-density lipoprotein

  •  
  • ISTD

    internal standard

  •  
  • LC

    liquid chromatography

  •  
  • LDL

    low-density lipoprotein

  •  
  • LSD

    lysosomal storage disorder

  •  
  • MPS

    mucopolysaccharidosis

  •  
  • MRM

    multiple-reaction monitoring

  •  
  • PC

    phosphatidylcholine

  •  
  • PG

    phosphatidylglycerol

  •  
  • PG

    C14:0/C14:0, 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

  •  
  • PG

    C18:1/C18:1, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]

  •  
  • VLDL

    very-low-density lipoprotein

References

References
1
Postle
A. D.
Heeley
E. L.
Wilton
D. C.
A comparison of the molecular species compositions of mammalian lung surfactant phospholipids
Comp. Biochem. Physiol. A Mol. Integr. Physiol.
2001
, vol. 
129
 (pg. 
65
-
73
)
2
Amidon
B.
Brown
A.
Waite
M.
Transacylase and phospholipases in the synthesis of bis(monoacylglycero)phosphate
Biochemistry
1996
, vol. 
35
 (pg. 
13995
-
14002
)
3
Heravi
J.
Waite
M.
Transacylase formation of bis(monoacylglycerol)phosphate
Biochim. Biophys. Acta
1999
, vol. 
1437
 (pg. 
277
-
286
)
4
Kobayashi
T.
Beuchat
M. H.
Chevallier
J.
Makino
A.
Mayran
N.
Escola
J. M.
Lebrand
C.
Cosson
P.
Gruenberg
J.
Separation and characterization of late endosomal membrane domains
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
32157
-
32164
)
5
Kolter
T.
Sandhoff
K.
Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids
Annu. Rev. Cell Dev. Biol.
2005
, vol. 
21
 (pg. 
81
-
103
)
6
Kobayashi
T.
Beuchat
M. H.
Lindsay
M.
Frias
S.
Palmiter
R. D.
Sakuraba
H.
Parton
R. G.
Gruenberg
J.
Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport
Nat. Cell Biol.
1999
, vol. 
1
 (pg. 
113
-
118
)
7
Wassall
S. R.
Brzustowicz
M. R.
Shaikh
S. R.
Cherezov
V.
Caffrey
M.
Stillwell
W.
Order from disorder, corralling cholesterol with chaotic lipids. The role of polyunsaturated lipids in membrane raft formation
Chem. Phys. Lipids
2004
, vol. 
132
 (pg. 
79
-
88
)
8
Walkley
S. U.
Secondary accumulation of gangliosides in lysosomal storage disorders
Semin. Cell Dev. Biol.
2004
, vol. 
15
 (pg. 
433
-
444
)
9
McGlynn
R.
Dobrenis
K.
Walkley
S. U.
Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders
J. Comp. Neurol.
2004
, vol. 
480
 (pg. 
415
-
426
)
10
Liour
S. S.
Jones
M. Z.
Suzuki
M.
Bieberich
E.
Yu
R. K.
Metabolic studies of glycosphingolipid accumulation in mucopolysaccharidosis IIID
Mol. Genet. Metab.
2001
, vol. 
72
 (pg. 
239
-
247
)
11
Hopwood
J. J.
Muller
V.
Harrison
J. R.
Carey
W. F.
Elliott
H.
Robertson
E. F.
Pollard
A. C.
Enzymatic diagnosis of the mucopolysaccharidoses: experience of 96 cases diagnosed in a five-year period
Med. J. Aust.
1982
, vol. 
1
 (pg. 
257
-
260
)
12
Lowry
O. H.
Rosebrough
N. J.
Farr
A. L.
Randall
R. J.
Protein measurement with the Folin phenol reagent
J. Biol. Chem.
1951
, vol. 
193
 (pg. 
265
-
275
)
13
Havel
R. J.
Eder
H. A.
Bragdon
J. H.
The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum
J. Clin. Invest.
1955
, vol. 
34
 (pg. 
1345
-
1353
)
14
Laulagnier
K.
Motta
C.
Hamdi
S.
Roy
S.
Fauvelle
F.
Pageaux
J.-F.
Kobayashi
T.
Salles
J.-P.
Perret
B.
Bonnerot
C.
Record
M.
Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization
Biochem. J.
2004
, vol. 
380
 (pg. 
161
-
171
)
15
Folch
J.
Lees
M.
Sloane Stanley
G. H.
A simple method for the isolation and purification of total lipids from animal tissues
J. Biol. Chem.
1957
, vol. 
226
 (pg. 
497
-
509
)
16
Han
X.
Yang
J.
Cheng
H.
Ye
H.
Gross
R. W.
Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry
Anal. Biochem.
2004
, vol. 
330
 (pg. 
317
-
331
)
17
Holbrook
P. G.
Pannell
L. K.
Murata
Y.
Daly
J. W.
Bis(monoacylglycero)phosphate from PC12 cells, a phospholipid that can comigrate with phosphatidic acid: molecular species analysis by fast atom bombardment mass spectrometry
Biochim. Biophys. Acta
1992
, vol. 
1125
 (pg. 
330
-
334
)
18
Kakela
R.
Somerharju
P.
Tyynela
J.
Analysis of phospholipid molecular species in brains from patients with infantile and juvenile neuronal-ceroid lipofuscinosis using liquid chromatography–electrospray ionization mass spectrometry
J. Neurochem.
2003
, vol. 
84
 (pg. 
1051
-
1065
)
19
Duffin
K.
Obukowicz
M.
Raz
A.
Shieh
J. J.
Electrospray/tandem mass spectrometry for quantitative analysis of lipid remodeling in essential fatty acid deficient mice
Anal. Biochem.
2000
, vol. 
279
 (pg. 
179
-
188
)
20
Besley
G. T.
Elleder
M.
Enzyme activities and phospholipid storage patterns in brain and spleen samples from Niemann–Pick disease variants: a comparison of neuropathic and non-neuropathic forms
J. Inherit. Metab. Dis.
1986
, vol. 
9
 (pg. 
59
-
71
)
21
Walkley
S. U.
Thrall
M. A.
Haskins
M. E.
Mitchell
T. W.
Wenger
D. A.
Brown
D. E.
Dial
S.
Seim
H.
Abnormal neuronal metabolism and storage in mucopolysaccharidosis type VI (Maroteaux–Lamy) disease
Neuropathol. Appl. Neurobiol.
2005
, vol. 
31
 (pg. 
536
-
544
)
22
Salvioli
R.
Tatti
M.
Ciaffoni
F.
Vaccaro
A. M.
Further studies on the reconstitution of glucosylceramidase activity by Sap C and anionic phospholipids
FEBS Lett.
2000
, vol. 
472
 (pg. 
17
-
21
)
23
Takahashi
Y.
Smith
J. D.
Cholesterol efflux to apolipoprotein AI involves endocytosis and resecretion in a calcium-dependent pathway
Proc. Natl. Acad. Sci. U.S.A.
1999
, vol. 
96
 (pg. 
11358
-
11363
)
24
Lewis
G. F.
Rader
D. J.
New insights into the regulation of HDL metabolism and reverse cholesterol transport
Circ. Res.
2005
, vol. 
96
 (pg. 
1221
-
1232
)
25
Arakawa
R.
Abe-Dohmae
S.
Asai
M.
Ito
J. I.
Yokoyama
S.
Involvement of caveolin-1 in cholesterol enrichment of high density lipoprotein during its assembly by apolipoprotein and THP-1 cells
J. Lipid Res.
2000
, vol. 
41
 (pg. 
1952
-
1962
)

Author notes

1

Present address: Baker Heart Research Institute, 75 Commercial Road, Melbourne, VIC 3006, Australia.

2

Present address: Department of Veterinary Preclinical Sciences, Faculty of Veterinary Science, University of Liverpool, Crown Street, Liverpool L69 7ZJ, U.K.