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Technical Briefs |
1 Clinical Laboratory Science, and 2 Protein Characterization & Modification, Genzyme Corporation, One Mountain Road, Framingham, MA 01701-9322
aauthor for correspondence: fax 508-820-7664, e-mail Crystal.Sung{at}genzyme.com
Fabry disease is a rare X-linked lysosomal storage disorder resulting from a deficiency in the 
-galactosidase A enzyme. Deficiency in the activity of this enzyme causes an accumulation of neutral glycosphingolipids, predominantly globotriaosylceramide (GL-3), in most nonneural tissues and in body fluids (1). Recent clinical studies indicate that tissue and plasma GL-3 concentrations in Fabry patients can be significantly reduced by enzyme replacement therapy (2)(3)(4)(5).
GL-3 (see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/) exists as a mixture of structural isoforms containing acyl chains ranging from 16 to 24 carbons in length with various degrees of saturation and hydroxylation. These variations make the extraction and quantification of GL-3 challenging (6)(7). Moreover, no well-characterized reference standards of known purity are available.
GL-3 in tissues and plasma has been measured by thin-layer chromatography (8)(9), liquid chromatography (LC) (10)(11)(12)(13)(14)(15), and gas chromatography(16), but the methods are labor-intensive and slow. An enzyme-linked immunosorbent assay (17) requires recombinant verotoxin B and polyclonal rabbit anti-verotoxin B. To date, the most rapid quantitative assays for total GL-3 have used flow-injection tandem mass spectrometry (MS/MS) (18)(19). We now report the development and use of a rapid LC/MS/MS method for the quantitative determination of total plasma GL-3.
Porcine GL-3 and porcine globotriaosylsphingosine A (lyso-GL-3), from Matreya, were 
98% pure by thin-layer chromatographic analysis. C16:0-GL-3 and C17:0-GL-3 were enzymatically synthesized from lyso-GL-3 at Genzyme Pharmaceuticals (18). C16:0-enriched GL-3 was prepared by gravimetrically combining C16:0-GL-3 and porcine GL-3 in a 9:25 g/g mass ratio. Methanol, water, and chloroform were HPLC grade.
Normal heparin-plasma samples from 104 men and 101 women were obtained from Interstate Blood Bank (Memphis, TN), ProMedDx LLC, and internal Genzyme sources. Heparin-plasma samples were also collected randomly from 57 Fabry patients enrolled in a clinical trial (3). Two sets of samples were collected: one set was from a group of patients who received intravenous FabrazymeTM from the onset of the trial, whereas the other set received placebo for 5 months and was then given Fabrazyme. All patients provided informed consent. The procedures were approved by the Institutional Review Boards and/or Ethics Committees of all participating centers. Pooled normal plasma (for method development and validation) was from 50 healthy donors. Quality-control (QC) materials were prepared by combining plasma from healthy donors or Fabry patients.
Briefly, 1.2 mL of chloroform-methanol, 60 µL of plasma, and 48 µL of 5 mg/L C17:0-GL-3 were pipetted (in the order listed) into a 2-mL microcentrifuge tube. The sample was subsequently extracted (20)(21) and purified by solid-phase extraction as described recently (6)(7). Purified samples were reconstituted with methanol (75 µL), vortex-mixed (30 s), and sonicated (3 min at 37 °C) before analysis by LC/MS/MS.
Plasma calibrants were prepared in bulk quantities based on previously reported procedures (6)(7). Purified calibrants were reconstituted with methanol and analyzed by LC/MS/MS as described above.
LC/MS/MS analyses were performed on a Waters Alliance HPLC separations module coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer operating in positive electrospray ionization mode.
Purified extracts were injected (20 µL) on two Luna C8 guard columns [8 x 3 mm (i.d.); 5 µm particle size] set at 45 °C and connected in series and were eluted (500 µL/min) with the gradient mobile phase conditions given in Table 1 of the online Data Supplement. Mobile phase A was 2 mmol/L ammonium acetate plus 1 mL/L formic acid in water, mobile phase B was 2 mmol/L ammonium acetate plus 1 mL/L formic acid in methanol, and mobile phase C was 2:1 (by volume) chloroform–methanol.
The MS/MS operating and detection conditions have been described in other reports (6)(7). Neutral loss scan spectra were collected for both plasma extracts and calibrators. The GL-3 isoforms were detected and summed based on the multiple-reaction monitoring transitions specified in Table 1
. The total GL-3 value (mg/L) was calculated by summing the responses from 10 individual isoforms (Fig. 2
, a and b, in the online Data Supplement).
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Commercial reference standards for human plasma GL-3 do not exist. Porcine GL-3 (Fig. 1A
) was found to be similar to human GL-3 (Fig. 1B
), but porcine GL-3 lacked the C16:0-GL-3 isoform usually found in high concentrations in human plasma GL-3. Porcine GL-3 was subsequently fortified with an appropriate amount of synthetic C16:0-GL-3 isoform to make the porcine material suitable for quantifying total GL-3 extracted from human plasma (Fig. 3a in the online Data Supplement). C17:0-GL-3 was also synthesized and used as an internal standard because this isoform is not a common isoform found in human plasma (Fig. 3b in the online Data Supplement).
The instrumental limit of detection for GL-3 was <0.01 mg/L (signal-to-noise ratio = 151). Repeat injection (n = 189) of a 5 mg/L GL-3 calibrator had area response and retention time CVs of 4.9% and 0.6%, respectively. The method limit of quantification was 0.5 mg/L with an injection imprecision of 15%. The bias in the determination at 0.5 mg/L was 14.7% (n = 10), and the measured value was different from the matrix signal with 97% confidence (Student t-test). The area response injection precision of QC materials was 
6% over 10 testing days.
Method accuracy was assessed based on 10 days of analysis. Measured results across the calibration range were within 94–114% of the expected values. The interassay imprecisio n (CV) of the calibration points (2–40 mg/L) was 6.0–14% (n = 10), whereas the intraassay imprecision of the points was 4.1% (n = 3). The solid-phase extraction procedure had a CV of 14% based on a 1-day analysis of 36 extractions performed by three operators over 2 months. The overall assay (including analyte extraction and LC/MS/MS analysis) had a CV of 17% based on 10 QC samples analyzed over a 2-month period.
The distributions of GL-3 concentrations in the plasma of 205 healthy donors and 57 Fabry patients are shown in Fig. 1C
. Healthy donors had a mean of 3.5 mg/L, a median of 3.3 mg/L, and a range of <2 to 11.2 mg/L for total GL-3. Fabry patients had a mean of 10.1 mg/L, a median of 9.4 mg/L, and a range of 3.6–16.7 mg/L for total GL-3. The difference between the healthy donors and Fabry patients was significant (P <0.0001) by the Student t-test. A decrease in plasma GL-3 concentration was observed in Fabrazyme-treated patients. In general, after 3 months of receiving Fabrazyme, plasma GL-3 concentrations were reduced to within the reference interval and essentially remained within the reference interval for the rest of the study (Fig. 4, a and b, in the online Data Supplement).
The first step in developing this method was to locate a well-characterized GL-3 reference standard for the preparation of GL-3 calibration curves. Unfortunately, at this time, no defined standards exist for the quantification of human plasma GL-3. In addition, the fatty acid composition of GL-3 varies depending on the species and the matrix of the source material (Fig. 1
, A and B). The C16-enriched GL-3 calibrator described here has been developed so that throughout the entire process of extraction, purification, and LC/MS/MS analysis, the total lipid profile of the calibrator would exhibit characteristics similar to human plasma GL-3. In addition to providing a tool for characterizing GL-3 isoforms (6)(7), LC/MS/MS also allows for the sensitive quantification of low concentrations of total GL-3. The GL-3 limit of quantification (0.5 mg/L) for this method, including extraction and purification, is well within the detection limits of the MS system.
Comparison of this new method with previously reported methods in terms of accuracy, reproducibility, and analyst effort is not straightforward. Differences involved in the multiple steps of the extraction methods, selection of Fabry patients and healthy donors, preparation and sources of calibrators, and principles of methodologies will contribute to the variances inherent to GL-3 determinations. Zeidner et al. (17) used an ELISA method to determine total GL-3; the mean plasma GL-3 concentrations in male and female Fabry heterozygotes were reported as 12.6 and 1.1 mg/L, respectively, whereas the concentration in healthy individuals was 0.9 mg/L. In contrast, Mills et al. (18) reported that the mean total GL-3 concentrations, measured by flow-injection MS/MS in Fabry and normal plasma, were 29.1 and 8.4 mg/L, respectively. Schiffmann et al. (10) reported mean plasma GL-3 values in Fabry (placebo and treated) of 10.96 and 12.14 nmol/mL (equivalent to 11.6 and 12.8 mg/L).
Longitudinal studies of biomarkers such as GL-3 require that the technique is reproducible. To ensure long-term reproducibility of the method, QC materials were used to evaluate the proficiencies of different operators and reagents and other key factors in the method. The reproducibility of the extraction and of the overall method demonstrates that the method is rugged for individual analysts and reagents, especially considering the overall diversity of GL-3 molecules, the complexity of a plasma matrix, and the number of steps involved in the analysis.
In summary, we have developed a rapid LC/MS/MS method to determine and monitor changes in the concentration of total GL-3 in human plasma. The method provides a sensitive, reliable, and reproducible technique to monitor GL-3 in patients with Fabry disease treated by various therapeutic modalities.
Acknowledgments
We acknowledge Carole Elbin and William Chung for their initial guidance in lipid extraction. In addition, Amy Cotsonas and Christian Braithwaite participated in the long-term reproducibility studies. We would also like to acknowledge Fei Wang, Jim MacDougall, and Biomedical Operations at Genzyme for contributions to the statistical analysis.
References
-Galactosidase A deficiency: Fabry disease. Scriver CR Beaudet AL Sly WS Valle D eds. The metabolic and molecular bases of inherited disease 8th ed 2001:3733-3774 McGraw-Hill New York. .
-galactosidase A: replacement therapy in Fabry’s disease. New Engl J Med 2001;345:9-16.-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease. Proc Natl Acad Sci U S A 2000;97:365-370.
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), and the right-hand y axis represents the frequency of Fabry patients (n = 57; 
)