Analytical Methods

2.2.4.1. Assay of total protein by the Bradford method

The concentration of protein in tissue extracts or cells lysate was measured by Bradford protein assay (Bradford, 1976, Compton and Jones, 1985). Concentrated standard stock solutions of bovine serum albumin (BSA) was calibrated by UV absorption spectrophotometry using the extinction coefficient at 279 nm for a 1% (10 mg/ml) solution; ε279 (1%) = 6.9 cm-1 (Peters, 1962). Protein samples were diluted in

the range 0.05 to 0.3 mg/ml. Test samples, BSA standards and blanks in triplicate (20 µl per well) were mixed with 200 µl of diluted Bradford reagent in a 96-well clear microplate. Absorbance at 595 nm was read 10 min after addition. The concentration of protein in test samples was deduced by interpolation of the calibration curve.

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2.2.4.2. Activity of glyoxalase 1

The activity of Glo1 was determined by measuring the initial rate of formation of S-D-lactoylglutathione from the MG-GSH hemithioacetal which is formed non-enzymatically from MG and reduced glutathione (GSH). The reaction was conveniently determined by following the increase in absorbance at 240 nm for which Δε240 = 2.86 mM−1 cm−1.

Hemithioacetal was prepared by pre-incubation of 2 mM MG with 2 mM GSH for 10 min in 50 mM sodium phosphate buffer, pH 6.6 and 37°C (980 μl). The tissue extract or cell lysate (20 μl) was added and the absorbance at 240 nm was monitored with time for 5 min. The activity of Glo1 is deduced from the initial increase in absorbance, corrected for homogenization buffer blank. Glo1 activity is given in units per mg protein where one unit of Glo1 activity is the amount of enzyme which catalyses the formation of 1 μmol SLG per min under assay conditions (Arai et al., 2014).

2.2.4.3. Activity of glyoxalase 2

The activity of Glo2 was determined by measuring the initial rate of hydrolysis of SLG to D-lactate and GSH, followed spectrophotometrically at 240 nm; Δε240 = - 3.10 mM-1cm-1 (Clelland and Thornalley, 1991, Allen et al., 1993).

SLG (0.3 mM) was incubated in 50 mM Tris/HCl, pH 7.4 at 37°C and the tissue extract, cell lysate or lysate buffer for the blank was added at a 50-fold dilution to a final volume of 1 ml. The reaction was monitored for absorbance at 240 nm for 5 min at 37°C. The initial rate of change in absorbance was deduced. One unit of Glo2 activity is the amount of enzyme which catalyses the hydrolysis of 1 µmol SLG per min under assay conditions (Allen et al., 1993).

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2.2.4.4. Assay of methylglyoxal reductase activity

The activity of MG reductase was determined by measuring the initial rate of reduction of MG to hydroxyacetone (major product) and lactaldehyde (minor

product), conveniently followed by measuring the rate of oxidation of NADPH, followed spectrophotometrically at 340 nm; Δε340 = 6.2 mM-1cm-1 (Murata et al.,

1985).

NADPH (0.1 mM) and MG (1 mM) was incubated in 50 mM sodium

phosphate buffer, pH 7.4 at 37°C and the tissue extract or lysate buffer for the blank was added to a final volume of 1 ml. The reaction monitored for absorbance at 340 nm for 5 min at 37°C. The initial rate of change in absorbance was deduced. One unit of MG reductase activity is the amount of enzyme which catalyses the

hydrolysis of 1 µmol of NADPH per minute under assay conditions (Murata et al., 1985).

2.2.4.5. Western Blot

Tissue protein extract or cell lysate (20 µg) was mixed with 4x Laemmli sample buffer and separated using 8–16% Mini-PROTEAN® TGX™ gel in Tris/glycine/SDS electrophoresis buffer. The separated proteins were transferred from the gel to nitrocellulose membrane by Trans-Blot® Turbo™ RTA midi

nitrocellulose transfer kit using PowerPac™ basic power supply electrophoresis unit (semi-dry transfer at 2.5 Ampere and 25 volt) for 3 min. The sandwich layer of semi- dry transfer consisted of filter paper, gel and membrane, immersed in transfer buffer. Membranes were blocked with 5% dried milk protein in Tris-buffered saline with tween-20 (TBS-T buffer; 500 mM NaCl, 20 mM Tris; pH 7.5 and 0.05% Tween-20) for 1 hour. Membranes were then probed with primary antibody using pre-

determined concentrations of anti-Glo1 antibody or ESCs markers including SOX2, NANOG and OCT4 overnight at 4ºC. After blotting with primary antibody, the membranes were washed with 3 x TBS-T buffer. Membranes were then probed with secondary antibody (anti-rabbit) at room temperature for 1 h. Membranes were rinsed with 3 x TBS-T and developed with ECL reagent and photographic films.

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Glo1 blotting results were normalised to β-actin (protein loading control).

Membranes were scanned and quantified with ImageQuant densitometry software.

2.2.4.6. Assay of D-lactate

The concentration of D-lactate in the media or tissue extract was assayed by endpoint enzymatic assay, modified for microplate techniques, with D-lactic dehydrogenase using fluorescence (McLellan et al., 1992).

Inclusion of hydrazine in the assay cocktail removes pyruvate from the equilibrium as pyruvyl-hydrazone and drives the forward reaction to completion and endpoint. The amount of D-lactate in the sample is deduced from the amount of NADH formed, determined by microplate fluorimetry with fluorescence detection at excitation wavelength 340 nm and emission wavelength 460 nm.

Media samples (500 µl) were deproteinsed with perchloric acid (PCA, 0.6 M; 1.0 ml), incubated on ice for 10 min, vortex-mixed and centrifuged (7000 g, 5 min, 4oC). The supernatant (700 µl) was neutralised with potassium bicarbonate (175 µl, 2 M) and centrifuged to sediment the resulting potassium perchlorate precipitate. For the tissue extracts, tissue samples (10 mg) were homogenized with PCA (0.6 M; 250 μl), incubated on ice for 10 min, vortex-mixed and centrifuged (4000 g, 5 min, 4o_C).

The supernatant (200 µl) was neutralised with potassium bicarbonate (80 µl, 2 M) and centrifuged to sediment the resulting potassium perchlorate precipitate. Solutions were placed in a centrifugal evaporator and vacuum applied (20 mmHg) to remove dissolves CO2 formed during the neutralisation. The neutralised PCA extract from

tissue or media (100 µl, pH 7) was assayed for D-lactate by incubating with glycine- hydrazine buffer (1.2 M glycine, 0.5 M hydrazine dihydrochloride, 2.5 mM

DETAPAC, pH 9.2; 100 μl), NAD+ _{(25 µl, 4 mM) and D-lactic dehydrogenase (25}

µl, 250 U/ml) for 2 h. Control samples were run in parallel without D-lactic

dehydrogenase. A calibration curve was constructed in the range 0 - 6 nmol D-lactate for media samples (Figure 2.9) and the curve was constructed in the range 0 - 3 nmol D-lactate for tissue samples.

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Figure 2.9: Typical calibration curve for assay of D-lactate. Linear regression

equation: Fluorescence (arbitrary units) = (6632 ± 508) x D-lactate (nmol); R2 = 0.995 (data are mean ± SD; n = 3).

2.2.4.7. Assay of L-lactate

The concentration of L-lactate in cell culture medium was determined similarly as the D-lactate method described in section 2.2.4.6. A standard curve was constructed using L-lactate standards in range 0 - 10 nmol and L-lactate

dehydrogenase was used instead of D-lactate dehydrogenase used for the D-lactate assay. Since the cellular levels of L-lactate are 50-100 fold higher than D-lactate, media samples were first diluted with water to ensure that concentrations measured were within the standard curve range. Figure 2.10 shows a calibration curve for L- lactate. 0 10000 20000 30000 40000 50000 0 2 4 6 Fluoresce nc e (A rbi tr ary uni ts) D-Lactate (nmol)

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Figure 2.10: Typical calibration curve for assay of L-lactate. Linear

regression equation: Fluorescence (arbitrary units) = (683 ± 87) x L-lactate (nmol); R2 = 0.998 (data are mean ± SD; n = 3).

2.2.4.8. Assay of D-glucose

The concentration of glucose in cell culture media was determined using an end-point enzymatic assay using a commercial assay reagent (containing 1.5 mM NAD+, 1 mM ATP, 1 unit/ml hexokinase and 1 unit/ml glucose-6-phosphate (G6P) dehydrogenase) and 1 mg/ml D-glucose standard. The enzymatic basis of the assay is illustrated in Figure 2.11.

Figure 2.11: The coupled enzyme reactions of the glucose assay.

The formation of NADH was measured by spectrophotometrically at 340 nm. Since equimolar amounts of glucose are phosphorylated to G6P and NAD+ _reduced

to NADH in this reaction, the increase in absorbance at 340 nm is directly

proportional to the concentration of glucose. A standard curve was constructed in the range of 0 - 1.5 mM D-glucose - Figure 2.12.

0 2000 4000 6000 8000 0 2 4 6 8 10 Fluoresce nc e (A rbi tr ary uni ts) L-Lactate (nmol)

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Figure 2.12: Typical calibration curve for assay of D-glucose. Linear

regression equation: A340 (A.U.) = (0.414 ± 0.0142) x D-glucose (mM); R2 = 0.994 (data are mean ± SD; n = 3).

Media samples at baseline and after treatment were collected and diluted with water appropriate for estimates to fall within the range of the calibration curve. Aliquot of standards and diluted samples (25 µl) was added to a clear 96-well plate with 225 µl of the assay reagent. The microplate was then incubated at room temperature for 15 min and absorbance was measured at 340 nm using FLUOstar optima microplate reader. The concentration of D-glucose was deduced from the standard curve and D-glucose levels were expressed in mM. The consumption of D- glucose was also calculated by subtracting the D-glucose levels after experimental incubations from baseline media concentrations and expressed in nmol/day/106 _cells.