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Technical Briefs |
1 Centre for Metabolic Disease, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium;
aaddress correspondence to this author at: Centre for Metabolic Disease, Campus Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium; fax 32-16-347284, e-mail hubert.carchon{at}med.kuleuven.ac.be
Congenital disorders of glycosylation (CDG) are a newly delineated group of inherited multisystem disorders associated with abnormal glycosylation of glycoproteins (1). In CDG group I, which includes all defects in N-glycan assembly (2), the subgroup CDG type Ib, attributable to phosphomannose isomerase (EC 5.3.1.8.) deficiency, is treatable by mannose supplementation (3). Monitoring of this treatment necessitates the availability of methods to quantify D-mannose in serum.
The determination of mannose in serum is hampered by the presence of an 
100-fold excess of glucose. Jolley et al. (4) used high-resolution liquid chromatography, whereas Aloia (5) used gas-liquid chromatography after treating the sera with glucose oxidase. Soyama (6) and Akazawa et al. (7) used enzymatic methods that involved treatment with glucose oxidase. In all of these studies, the presence or elimination of glucose remained critical. Pitkänen and Kanninen (8) were able to measure mannose using gas chromatography-mass spectrometry. However, this method is not suitable for routine purposes. The assay proposed by Etchison and Freeze (9) involves the elimination of glucose by glucokinase (EC 2.7.1.2), followed by the removal of anionic products by a subtle ion-exchange chromatography step. Finally, the mannose concentration is determined enzymatically.
We investigated whether capillary electrophoresis (CE) of fluorophore-labeled carbohydrates was an appropriate method. In the resulting procedure, D-mannose can be determined in small amounts of serum in the presence of D-glucose without loss of selectivity, accuracy, and sensitivity.
1-Aminopyrene-3,6,8-trisulfonate (APTS) was purchased from Lambda Fluoreszenz Technologie GmbH. Sodium cyanoborohydride (1.0 mol/L in tetrahydrofuran) was obtained from Aldrich Chemical. Boric acid and ethanol were from Merck; glucokinase and the carbohydrates were obtained from Sigma. All reagents used were of analytical grade. D-Mannose, L-rhamnose, and D-glucose are hereafter referred to as mannose, rhamnose, and glucose in the text.
CE separations were performed in fused-silica capillaries from Polymicro Technologies [20 cm effective length x 20 µm (i.d.)] at 20 °C on a Beckman Instruments P/ACE 5000 system and monitored on column with a Beckman laser-induced fluorescence detector using an argon-ion laser with excitation at 488 nm and emission at 520 nm. Borate-NaOH (135 mmol/L, pH 10.2) was used as the running buffer (10). After a long or overnight period of inactivity, the capillary was washed sequentially with 0.1 mol/L NaOH, water, 10 mmol/L EDTA, water, and 1 mol/L phosphoric acid for 2 min and finally with water for 5 min. Before each analysis, the capillary was reconditioned for 3 min with running buffer followed by a sample injection for 3 s corresponding to a sample volume of 0.15 nL (0.2% of the effective capillary volume). Finally, water was injected for 1 s to rinse the capillary end and the electrode. Separation was performed at 25 kV for 10 min. At the end of each analysis, the capillary was rinsed with water, 1 mol/L phosphoric acid, and water for 30 s, 3 min and 1 min, respectively. Solutions were filtered through Millipore filters (0.45 nm membrane) before analysis. The rinsing steps before and between analyses to obtain a reproducible electroendosmotic flow are an essential part of the separation procedure.
Cold ethanol (90 µL; -20 °C) was added to a mixture of 5 µL of 1 mmol/L rhamnose and 25 µL of serum. After 30 min at -20 °C, samples were centrifuged at 13 000g (15 min; 4 °C), and the supernatant was evaporated. The derivatization procedure was essentially adapted from the method first described by Jackson (11) and discussed by Guttman et al. (12). Briefly, 2 µL of 0.1 mol/L APTS and 0.6 mol/L citric acid (13) in water and 2 µL of 1.0 mol/L sodium cyanoborohydride in tetrahydrofuran were added to the dried sample. The reductive amination was allowed to proceed for 90 min at 55 °C. Finally, the mixture was diluted with water to 400 µL for CE analysis. Data are represented by their estimated value ± SE.
The migration process in CE analysis, using fused-silica capillaries at alkaline pH, is dominated by the velocity of the electroendosmotic flow. Therefore, constant migration times require stable and reproducible electroendosmotic flows. By adding a rinsing step with 1 mol/L phosphoric acid to the separation procedure, we reduced the CV of the migration time (MT) for mannose to 2.5% (n = 13). Residual variation of the MT was further decreased by expressing the MT of the analytes in terms of the MT of the internal standard (IS). Consequently, the CV of mannose was further reduced to 0.07%. This relative migration time was therefore used as an identification parameter.
For the determination of mannose, rhamnose can be used as IS because both compounds are well separated (Fig. 1A
) and serum does not contain contaminating analytes. In the presence of 5 mmol/L glucose, mannose remains completely separated in the range from 5 to at least 500 µmol/L. Under the present assay conditions, the lower detection limit for mannose can be defined at 2.5 µmol/L.
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A linear relationship was obtained between mannose at concentrations of 5–500 µmol/L, in the presence of 200 µmol/L IS and 5 mmol/L glucose, and the corresponding peak area expressed relative to the peak area of the IS. For n = 12, the intercept, slope, and correlation coefficient were -0.003 ± 0.006, 0.005 ± 0.00003, and 0.999 ± 0.015, respectively.
The recovery of mannose added to the sample at different concentrations is shown in Table 1
. The results indicate that the protein precipitation step does not significantly alter the mannose concentration.
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The separation of mannose and glucose in the calibration mixtures indicated that no preliminary elimination of glucose was required. Fig. 1B
shows the electropherogram after glucose elimination according to the method of Etchison and Freeze (9). Glucose is largely removed but not completely absent. The mannose concentrations in this sample with and without glucose elimination were 44.7 ± 0.2 and 40.2 ± 0.3 µmol/L, respectively. The glucose concentration obtained after glucose elimination was 20.4 ± 0.5 µmol/L. A small but significantly higher mannose concentration was found when the glucose elimination procedure was included (n = 5; P < 0.001). We determined the serum mannose concentration in 26 pediatric controls and obtained a mean concentration of 30 ± 10 µmol/L (range, 7–45 µmol/L), whereas Panneerselvam et al. (14), using the enzymatic procedure, reported a mean mannose concentration of 54.5 ± 14.5 µmol/L (n = 32).
The electropherogram obtained during CE analysis is very simple and reflects the selectivity of the derivatization step. The number of reducing carbohydrates in serum seems to be limited to mannose and glucose, but we do not know why. Fructose, which immediately follows glucose in the electropherogram and is present in serum at 20 µmol/L (8), cannot be detected under present assay conditions. If the sample size is increased, care must be taken that the amount of APTS in the derivatization mixture is also adjusted.
Acknowledgments
The present work was supported by the Funds for Scientific Research-Flanders (Grant G.0305.98).
References
The following articles in journals at HighWire Press have cited this article:
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  T. Taguchi, I. Miwa, T. Mizutani, H. Nakajima, Y. Fukumura, I. Kobayashi, M. Yabuuchi, and I. Miwa Determination of D-Mannose in Plasma by HPLC Clin. Chem., January 1, 2003; 49(1): 181 - 183. [Full Text] [PDF]
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