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Clinical Chemistry 53: 742-747, 2007. First published March 1, 2007; 10.1373/clinchem.2006.079012
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(Clinical Chemistry. 2007;53:742-747.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

HPLC for Simultaneous Quantification of Total Ceramide, Glucosylceramide, and Ceramide Trihexoside Concentrations in Plasma

Johanna E.M. Groener1,a, Ben J.H.M. Poorthuis1, Sijmen Kuiper1, Mariette T.J. Helmond1, Carla E.M. Hollak2 and Johannes M.F.G. Aerts1

Departments of1 Medical Biochemistry and 2 Internal Medicine, University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands.

aAddress correspondence to this author at: Department of Medical Biochemistry, University of Amsterdam, Academic Medical Center, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Fax 31-20-6915519; e-mail j.e.groener{at}amc.uva.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Simple, reproducible assays are needed for the quantification of sphingolipids, ceramide (Cer), and sphingoid bases. We developed an HPLC method for simultaneous quantification of total plasma concentrations of Cer, glucosylceramide (GlcCer), and ceramide trihexoside (CTH).

Methods: After addition of sphinganine as internal calibrator, we extracted lipids from 50 µL plasma. We deacylated Cer and glycosphingolipids by use of microwave-assisted hydrolysis in methanolic NaOH, followed by derivatization of the liberated amino-group with o-phthaldialdehyde. We separated the derivatized sphingoid bases and lysoglycosphingolipids by HPLC on a C18 reversed-phase column with a methanol/water mobile phase (88:12, vol/vol) and quantified them by use of a fluorescence detector at {lambda}ex 340 nm and {lambda}em 435 nm.

Results: Optimal conditions in the Solids/Moisture System SAM-155 microwave oven (CEM Corp.) for the complete deacylation of Cer and neutral glycosphingolipids without decomposition were 60 min at 85% power, fan setting 7. Intra- and interassay CVs were <4% and <14%, respectively, and recovery rates were 87%–113%. The limit of quantification was 2 pmol (0.1 pmol on column), and the method was linear over the interval of 2–200 µL plasma. In samples from 40 healthy individuals, mean (SD) concentrations were 9.0 (2.3) µmol/L for Cer, 6.3 (1.9) µmol/L for GlcCer, and 1.7 (0.5) µmol/L for CTH. Plasma concentrations of GlcCer were higher in Gaucher disease patient samples and of CTH in Fabry disease patient samples.

Conclusions: HPLC enables quantification of total Cer, GlcCer, and CTH in plasma and is useful for the follow-up of patients on therapy for Gaucher or Fabry disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sphingolipidoses are inherited lysosomal storage disorders caused by lysosomal hydrolase deficiency leading to storage of specific glycosphingolipids. Two forms of therapies are available. In enzyme replacement therapy (ERT),1 the missing enzyme is administered to the patient intravenously. The enzyme is taken up by cells by receptor-mediated endocytosis, which leads to reduction of the stored material. Currently, this therapy is used in the treatment of Gaucher disease type 1 (1)(2) and Fabry disease (3)(4). Treatment of Niemann-Pick type B is under development. In substrate reduction therapy (SRT), the synthesis of glycosphingolipids is reduced by partial inhibition of glucosylceramide (GlcCer) synthase by deoxynojirimycin analogs (5), leading to reduction of the glycosphingolipid pool and eventually to reduction of the substrate load for lysosomal degradation by sphingolipid hydrolases. This therapy is applied to a subgroup of patients with Gaucher disease type 1 for whom ERT is not available (6). Because SRT is nonspecific, it could in principle be applied to all sphingolipidoses in which the degradation of GlcCer-derived sphingolipids is impaired. In contrast to the enzymes administered in ERT, the drug used for SRT can cross the blood–brain barrier and reach target cells in the brain and thus could be used in the treatment of sphingolipidosis with involvement of the central nervous system (7). SRT trials are under way for Niemann-Pick type C, juvenile GM2-gangliosidosis, and Gaucher disease type 3.

To monitor the effect of these therapies, glycosphingolipids in plasma must be measured. In addition to monitoring the primary storage products, it is important to measure possible changes in ceramide (Cer) concentrations and other intermediates or products in glycosphingolipid metabolism, particularly in SRT. Cer is an intermediate in synthesis of glycosphingolipid, sphingomyelin, sphingosine, and Cer-1-P, compounds that are increasingly recognized as mediators of cellular processes. They participate in various cell-surface–related processes such as cell differentiation, transmembrane signaling, cell recognition, and toxin binding (8)(9).

Several techniques are available to measure Cer and, in particular, neutral glycosphingolipids. These techniques include thin-layer chromatography with densitometry or immunochemical detection to HPLC and mass spectrometry (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Most of these methods, however, do not allow the simultaneous measurement of Cer and neutral glycolipids, or they require extensive calibration and expensive equipment (as in the case of mass spectrometry). Here we describe a simple method for the simultaneous quantification of Cer and neutral glycosphingolipids in plasma and its application for monitoring GlcCer and Cer in patients with Gaucher disease on ERT and SRT and for ceramide trihexoside (CTH) and Cer in patients with Fabry disease on ERT. The method is based on the conversion of glycosphingolipids and Cer to lysoglycosphingolipids and sphingoid bases by microwave-mediated saponification, as pioneered by Taketomi et al. (22) and discussed by Itonori et al. (23). We optimized this deacylation procedure and quantified the sphingoid bases and lysoglycosphingolipids after derivatization with o-phthaldialdehyde (OPA) by HPLC with fluorometric detection (24).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
We obtained C18-sphinganine, sphingosine, lyso-GlcCer, lyso-Cer, lyso-CTH, Cer, GlcCer, lactosylceramide, and CTH from Avanti Polar Lipids; methanol pro analysi and chloroform pro analysi from Merck KGaA; and OPA from Sigma.

plasma samples
We obtained samples from patients referred to the Lysosomal Outpatient Clinic of the Academic Medical Center in Amsterdam for assessment of severity of disease or institution of ERT or SRT. Control samples were obtained from healthy volunteers. Informed consent was provided according to the Declaration of Helsinki. Plasma samples were stored at –20 °C until use.

microwave oven
We used the Solids/Moisture System SAM-155 (CEM Corp.) with a power range of 0% to 100% of full power (630 W), an exhaust fan, and a rotating Teflon tray with 36 tube holes.

sample preparation and hplc
We added 1 nmol C18-sphinganine, as the internal calibrator, to 50 µL plasma. Lipids were extracted with 2 mL chloroform/methanol (1:1, vol/vol) for 30 min at room temperature, followed by centrifugation for 5 min at 1500g. We added 1 mL chloroform and 0.75 mL 0.73% NaCl to the clear extract to obtain phase separation according to Folch et al. (25) and removed the lower chloroform layer. We washed the upper phase once with 1 mL chloroform, and the combined chloroform layers were thoroughly evaporated to dryness. Using 0.5 mL freshly prepared 0.1 mol/L NaOH in methanol, we deacylated the lipids in 12 x 100 mm borosilicate glass tubes (Schott GL14) with polytetrafluorethylene-lined screw caps in the CEM microwave oven. For reasons of reproducibility and safety, 36 tubes filled with 0.5 mL of 0.1 mol/L NaOH in methanol were always present. To maximize deacylation and minimize decomposition, the optimal condition for this microwave oven proved to be an exposure time of 60 min at 85% power and a fixed setting of 7 for the exhaust fan (see Results).

After hydrolysis, the samples were cooled to room temperature inside the microwave oven. We transferred 50 µL of the deacylated lipid mixture into autosampler vials and derivatized the lipids for 30 min with the addition of 25 µL OPA reagent [5 mg OPA, 0.1 mL ethanol, 5 µL 2-mercaptoethanol, and 10 mL 3% (wt/vol) boric acid adjusted to pH 9.0], essentially as described by Merrill et al. (24). The final pH of the mixture during OPA derivatization was pH 10.7. We separated OPA-derivatized sphingoid bases and lysoglycosphingolipids by use of a Waters HPLC system consisting of a 600 Controller, 717plus Autosampler, and 474 Scanning Fluorescence Detector with an Altima BDS C18 3µ, 150 x 4.6 mm reversed-phase column. The mobile phase was methanol/water 88:12 (vol/vol). We quantified the OPA-derived lysocompounds with a fluorescence detector at {lambda}ex 340 nm and {lambda}em 435 nm. Peak identification was based on comparison of the retention times with those of authentic calibrators of the lysocompounds (sphingosine, sphinganine, lyso-GlcCer, and lyso-CTH) and those of Cer, GlcCer, and CTH after microwave-assisted deacylation. Peak integration was performed with Waters Millennium software. All samples were run in duplicate, and 2 reference samples were included in every run.

quantification, recovery, and imprecision studies
We used C18-sphinganine as the internal calibrator for quantification. The limit of detection was defined as the amount of sphinganine producing a signal-to-background ratio of 3. Plasma normally contains very low concentrations of sphinganine, which do not affect the quantification (see Results). We measured recovery by adding known amounts of Cer, GlcCer, and CTH to 2 different plasma specimens. Because Cer, GlcCer, and CTH consist of a mixture of different molecular species, we prepared calibrator solutions of these compounds used for the recovery studies using an estimated average molecular weight and exactly measured each concentration by quantification after deacylation. Recoveries were calculated for each concentration. To calculate within-assay imprecision, we measured plasma concentrations 10 times in the same assay series; between-assay imprecision was calculated using results from the same control and Fabry plasma samples analyzed on 10 different days. We checked the linearity of the method by serial 25-fold dilution of a Gaucher plasma and a Fabry plasma sample in phosphate-buffered saline in the lower concentration interval, and a 4-times-higher volume of plasma than used in the standard assay in the higher concentration interval. We defined the limit of quantification of Cer, GlcCer, and CTH with the current method as the amount that gave a signal-to-noise ratio of 5.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
microwave-assisted deacylation
Because our goal was to develop an assay suitable for a large set of samples, we selected an industrial microwave oven (CEM Solids/Moisture System SAM-155) with a rotating tray with 36 tube holes. We first determined the optimal microwave conditions—i.e., conditions for complete deacylation with minimal decomposition—for the alkaline hydrolysis of Cer and GlcCer in lipid extracts of plasma. Lipid extracts were redissolved in 0.5 mL of 0.1 mol/L NaOH in methanol, placed in the microwave oven, and exposed to different levels of energy for different time periods. Fig. 1 shows the effect of time and energy output on the hydrolysis of Cer and GlcCer at 2 different fan settings. At settings 6 and 7, hydrolysis time was set at 30, 60, and 90 min and the power was set at 75%, 85%, and 95%. At the higher setting of the fan (Fig. 1, A and B ), deacylation of Cer was complete after 30 min (Fig. 1A ) and did not vary significantly among the 3 power settings and 3 time periods. GlcCer deacylation (Fig. 1B ) was optimal at 85% and 95% power but was clearly lower at 75% power. The curve also shows that the optimal time for deacylation of GlcCer was between 60 and 120 min; at shorter time periods, deacylation was incomplete. At a lower fan setting of 6, which results in a higher temperature and overall higher energy input, a decrease in the amount of sphingosine and lyso-GlcCer was noted at longer time periods (Fig. 1, C and D ), suggesting decomposition of the (glyco)sphingolipids. Therefore 60 min at 85% power and fan setting 7 was selected as the optimal condition for the alkaline hydrolysis of Cer and GlcCer. The settings of the microwave oven for the optimal hydrolysis of CTH were found to be the same as for GlcCer (data not shown). It should be emphasized that these settings are specific for the CEM Solids/Moisture System SAM-155 microwave oven, filled with 36 Schott GL14 tubes containing 0.5 mL 0.1 mol/L NaOH in methanol.


Figure 1
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  		Figure 1. Time course of the formation of sphingosine and lyso-GlcCer from Cer and GlcCer in plasma at different fan and power settings.

(A, B), fan setting 7. (C, D), fan setting 6. (A, C), formation of sphingosine from Cer at different power settings. (B, D), formation of lyso-GlcCer from GlcCer at different power settings.

The chromatograms for plasma from a control and patients with Gaucher and Fabry disease are shown in Fig. 2 . The chromatograms show small unidentified peaks that do not interfere with those of the (glyco)sphingolipids.


Figure 2
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  		Figure 2. Example HPLC chromatograms of plasma from a control, a patient with Gaucher disease, and a patient with Fabry disease.

Peak identification: internal calibrator (IS), sphinganine; 1, sphingosine; 2, lyso-GlcCer; 3, tentative lysolactosylceramide; 4, lyso-CTH.

Sphingoid bases and lysoglycosphingolipids are quantified with this method, so it is important to know the amount of lysoglycosphingolipids and sphingoid bases originally present in the plasma samples. To measure these, we applied lipid extracts to the column without deacylation and with and without the addition of the internal calibrator sphinganine. We found no detectable amounts of lysoglycosphingolipids. We detected very low concentrations of sphingosine, <3% of the sphingosine present after deacylation of Cer and sphinganine. The amount of sphinganine was <2% of the amount of the internal calibrator.

validation of the method
The limit of detection achieved with calibrators applied directly to the HPLC, defined as peak signal-to-noise ratio >3, was 20 fmol. Routinely, only 2% to 5% of the extracted glycolipids were injected onto the HPLC column. The limit of quantification obtained with serial dilution of plasma, defined as signal-to-noise ratio of 5, was 2 pmol present in the lipid extract (0.1 pmol on the column). In practice, 2 µL plasma gave a signal-to-noise ratio >5 for all components measured.

We measured recoveries of Cer, GlcCer, and CTH by standard addition of these lipids to plasmas. The amounts of Cer, GlcCer, and CTH showed a linear function of the amounts added, and the recoveries were 87% to 113% (Table 1 ).


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  		Table 1. Recovery studies.1

We tested the linearity of Cer, GlcCer, and CTH measurement after serial dilution of a plasma sample from a Gaucher patient with high GlcCer concentrations and a plasma sample from a Fabry patient with high CTH concentrations. In the Gaucher plasma, the concentrations were Cer 6.49, GlcCer 20.74, and CTH 1.52 µmol/L. In the Fabry plasma, the concentrations were Cer 7.22, GlcCer 6.20, and CTH 7.90 µmol/L. The amount of lipids measured was linear with the volume of plasma over the whole interval between 2 and 200 µL. For individual lipids, the correlation coefficient was >0.99 (Fig. 3 ).


Figure 3
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  		Figure 3. Linearity of the measurement of Cer, GlcCer, and CTH in plasma ranging in volume from 2 to 200 µL.

(A), Gaucher plasma: filled diamonds, Cer (R2 = 0.9967); open squares, GlcCer (R2 = 0.9934); filled triangles, CTH (R2 = 0.9888). B, Fabry plasma: filled diamonds, Cer (R2 = 0.9994); open squares, GlcCer (R2 = 0.9996); filled triangles, CTH (R2 = 0.9987).

The intraassay CV (n = 10) measured in Gaucher plasma and Fabry plasma was <4% for Cer, GlcCer, and CTH. The interassay CV (n = 10) was <14%.

application of the method
To assess the suitability of this method for the follow-up of Gaucher and Fabry patients during therapy, we investigated samples from 4 Gaucher patients, 2 on ERT and 2 on SDT, and 2 hemizygous Fabry patients on ERT. We compared the concentrations in plasma of the patients before therapy (Fig. 4 , t = 0) with those of plasma from 40 controls. Before start of therapy, GlcCer was higher in patients with Gaucher disease than in controls [mean concentration of GlcCer in plasma from controls was 6.3 (1.9) µmol/L (n = 40)]. On therapy, GlcCer decreased. Cer concentrations were within the reference interval [mean concentration of Cer in plasma from controls was 9.0 (2.3) µmol/L (n = 40)]. No change in Cer concentrations was noticed during the 24 months of therapy, either ERT or SRT (Fig. 4 ). In patients with Fabry disease, increased concentrations of CTH were measured before therapy (t = 0 in Fig. 4 ) [mean concentration of CTH in plasma from controls was 1.7 (0.5) µmol/L (n = 40)]. On therapy, the concentrations of CTH in plasma of Fabry patients decreased (Fig. 4 ).


Figure 4
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  		Figure 4. Effect of therapeutic intervention on plasma GlcCer and Cer concentrations in Gaucher patients and on CTH and Cer concentrations in Fabry patients.

(A, B), Gaucher patients 1 and 2 on ERT. C and D, Gaucher patients 3 and 4 on SRT: filled circles, GlcCer; open circles, Cer. E and F, Fabry patients 1 and 2 on ERT: open squares, CTH; filled squares, Cer.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed an HPLC method for simultaneously quantifying Cer and neutral glycosphingolipids in large numbers of plasma samples. The method is based on the microwave-assisted hydrolysis of glycosphingolipids and Cer (22)(23). By critically examining the efficiency of the microwave-assisted hydrolysis of Cer, GlcCer, and CTH, we established optimal conditions for deacylation. The procedure depends on achieving complete and reproducible deacylation of glycosphingolipids and Cer without decomposition. Because we were interested in a method suitable for measurements of large numbers of samples, we used a laboratory-grade instrument supplied with a Teflon tray suitable for 36 tubes. The power setting, fan setting, and time are optimal for this microwave oven. In addition, the oven should always be filled with 36 tubes containing 0.5 mL of 0.1 mol/L NaOH in methanol. Optimal conditions for deacylation would need to be established for different microwave ovens.

We show the effect of therapy on the concentrations of GlcCer and Cer in plasma from 4 Gaucher disease patients. Our data for plasma GlcCer in plasma from controls and Gaucher patients compared well with those obtained with thin-layer chromatography and electrospray ionization–tandem mass spectrometry (26)(27)(28)(29)(30). In the 4 patients, GlcCer concentrations decreased during both ERT and SRT. In the 2 Fabry patients, concentrations of CTH decreased upon therapy, as has been reported (13)(30)(31). These data illustrate the suitability of the new method for the follow-up of patients with Gaucher and Fabry disease during therapy. Data on Cer concentrations in human plasma are scarce. Our data are comparable to those published (32)(33)(34).

We obtained total concentrations of Cer and the glycosphingolipids of interest, independent of their molecular fatty acid composition. Recently, mass spectrometric analysis of sphingolipids has been described (16)(17)(18)(19)(20)(21). Mass spectrometric analysis renders information and quantification of molecular species of a particular glycosphingolipid; however, quantification is more difficult since each sample should ideally be supplemented with appropriate calibrators for each molecular form. In addition, mass spectrometry requires expensive equipment and specialized expertise, which are not always available in clinical chemistry laboratories. Our HPLC fluorescence detection method provides an alternative approach for simultaneous quantification of neutral glycosphingolipids and Cer in plasma and can be used to monitor plasma concentrations of Cer, GlcCer, and CTH in patients with Gaucher disease and Fabry disease during therapy.


   Footnotes
 
1 Nonstandard abbreviations: ERT, enzyme replacement therapy; SRT, substrate reduction therapy; GlcCer, glucosylceramide; Cer, ceramide; CTH, ceramide trihexoside; OPA, o-phthaldialdehyde.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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