Hexosamine pathway intermediate assay

6.5.1

Aim and principle

This assay was designed to separate by HPLC and quantify hexosamine biosynthesis path- way intermediates by UV absorbance. Specifically, the assay quantified levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucose (UDP- Glc). Samples were initially prepared by anion exchange solid phase extraction before sep- aration on an anion exchange HPLC column. Guanosine 5'-diphospho-_d-mannose (GDP- mannose) was chosen as an internal standard due to its low abundance in human cells and tissues.

This method was based on the work initially validated by Robinson et al. [1995] who adapted an original assay developed in 1985 [Wice et al., 1985]. This method was improved herein by eliminating the need for ozone-depleting solvents and reducing spurious peaks present on the chromatogram by adding solid phase extraction based on previous work by Span et al. [2001]. Other methods (e.g. capillary zone electrophoresis [Lehmann et al., 2000]) have been published to determine the concentration of these metabolites but HPLC was chosen due to equipment availability and prior experience with the technique.

6.5.2

Standard preparation

Authentic standards for the assay were calibrated and used to prepare standard curves for sample quantitation. Both UDP-Glc and UDP-GlcNAc were prepared in 10 mM KH2PO4 at pH 7.0 before spectrophotometric calibration at 262 nm (ε262 =10 000 M−1cm−1 at pH 7.0 [Dawson et al., 1986]). For standard use, a mixture of UDP-Glc and UDP-GlcNAc was prepared with both analytes at a concentration of 150µM. Standards were prepared as described in Table 6.8. Each of the standards was subjected to solid phase extraction in a manner identical to that of the samples.

6.5.3

Sample preparation

Cells were grown in each test medium for five days. Cultured cells close to confluence from one T-175 tissue culture flask (∼7.5×106 _{cells) were removed by trypsinisisation} and sedimented by centrifugation (5 min, 125×g, 4℃) for each sample. The supernatant was discarded and the cell pellet washed by resuspension in ice-cold PBS followed by re-sedimentation. Ice-cold lysis buffer comprising 100 mM KCl, 1 mM EDTA and 50 mM

KH2PO4 at pH 7.5 (1 mL) was added to each cell pellet. All steps from this point forward were performed on ice.

The cells in the pellet were lysed by sonication (10 s, 75 W) and the membranes clarified from the lysate by centrifugation (30 min, 20 000×g, 4℃). The membrane pellet was discarded and the supernatant collected. 50µL of the supernatant was reserved for protein assay and 700µL was used for the remaining assay steps.

Each 700µL supernatant aliquot was mixed with 700µL 1.2 M perchloric acid to precipitate protein and 10µL 1 mM GDP-mannose (10 nmol) added as an internal standard. The samples were mixed by brief vortexing and allowed to stand on ice for 10 min to allow complete precipitation. The protein precipitate was sedimented by centrifugation (10 min, 13 500×g, 4℃) and the supernatant reserved (approximately 1.3 mL supernatant was re- covered in each case). 10 times the volume of each supernatant of KH2PO4 at pH 2.5 was added to each supernatant before beginning solid phase extraction.

6.5.4

Solid phase extraction

The solid phase extraction (SPE) used Supelclean LC-SAX anion exchange cartridges (3 mL capacity, 500 mg packing material each). A vacuum manifold was used to speed up sample permeation through the packing material but for optimal binding of analytes to the sub- strate, the flow rate was never allowed to exceed 5 mL min−1_{. Prior to use the columns were} conditioned by applying the following to each cartridge in turn, in each case leaving 0.5– 1 mm of solvent on top of the frit after each application: 2 mL methanol, 2 mL water, 2 mL 10 mM KH2PO4 at pH 2.5. After conditioning, the samples were loaded onto and drawn through the SPE cartridges. The cartridges were washed with 5×1 mL 10 mM KH2PO4at pH 2.5 followed by 2.5 mL 50 mM KH2PO4 at pH 2.5 and finally 0.5 mL 150 mM KH2PO4

Standard (nmol) 1 mM GDP-mannose (µL) 150µM standard mix (µL) 10 mM KH2PO4 at pH 2.5 (mL) 1 10 6.67 12.98 5 10 33.33 12.96 10 10 66.67 12.92 20 10 133.3 12.86 35 10 233.3 12.76 50 10 333.3 12.66

at pH 7.5. All eluates were discarded. The compounds of interest were eluted using 2 ×

0.75 mL 150 mM KH2PO4at pH 7.5. The tubes were left to run dry and the entire volume of eluate was collected. The samples were spin-filtered (0.22µm pore size, 10 min, 4000×g, 4℃) and transferred to HPLC vials ready for injection. Samples were stored at 4℃ prior to analysis.

6.5.5

HPLC conditions

The HPLC column used was a 200x4.6 mm HEMA-IEC Bio-Q anion exchange column with a 10µm particle size. The mobile phases were 15 mM ammonium formate, pH 3.8 (A) and 1 M ammonium formate, pH 4.5 (B). Water and 30% IPA were used for rinsing salt from the system and cleaning the column after sample runs. The column was held in a thermostatic column oven at 25℃. The mobile phase increased from 0% to 50% B over 25 min using a concave gradient (slope 8 in the Chromeleon software) and is shown in Figure 6.10. The mobile phase was held at 50% B for 2.5 min before returning to 100% A for equilibration (7.5 min). The flow rate was 2 mL min−1 _{and detection was by UV absorbance at 262 nm.} The autosampler was thermostatically controlled at 4℃and the injection volume was 100µL. The needle was washed before every injection using water. The column was washed with 100% B prior to 30% IPA in water. An example chromatogram is given in Figure 6.11. Example standard curves generated using the peak area ratio of the analyte peak/internal standard peak are given in Figures 6.12 and 6.13. Analyte recoveries after SPE are given in Table 6.9.

Analyte Recovery (%)

UDP-Glc 90.1%

UDP-GlcNAc 86.4%

GDP-Man 75.4%

Figure 6.10: Hexosamine pathway intermediate assay mobile phase gradient

Figure 6.11: Hexosamine pathway intermediate assay example chromatogram. This chro- matogram shows peaks for UDP-Glucose and UDP-N-acetylglucosamine (5 nmol each) as well as the internal standard, GDP-mannose (10 nmol).

Figure 6.12: Example standard curve for UDP-glucose analysis. This is a typical standard curve in the range of 1 to 50 nmol.

Figure 6.13: Example standard curve for UDP-N-acetylglucosamine analysis. This is a typical standard curve in the range of 1 to 50 nmol.