**6. Instrument calibration**

240 Bioenergetics

The components (panel A, left to right) are circulating water bath, power supply for the mixer, sample chamber (panel B) attached to PMT, high voltage power supply for PMT, computer with PCI-DAS board, and monitor with developed software running. The PMT is connected to PCI-DAS board on the

Fig. 2. A sealed reaction vial containing Pd phosphor solution, stirring bar and a mouse liver

back of the computer. The developed software program interface is shown in panel C.

Fig. 1. The data acquisition system and developed software program.

specimen.

The instrument was calibrated with -glucose and glucose oxidase system.


The reaction contained PBS, 3 M Pd phosphor, 0.5% fat-free albumin, 50 g/mL glucose oxidase and various concentrations of -glucose. To achieve a high signal-to-nose ratio throughout the entire range of [O2], the photomultiplier tube was operated at 450 volts. Representative pulses (with exponential fits) for reactions containing PBS with 0, 125 or 500 M -glucose, 50 g/mL glucose oxidase, 3 M Pd phosphor and 0.5% fat-free bovine serum albumin are shown in Fig. 3.

Fig. 3. Representative phosphorescence pulses for reaction mixtures containing 0, 125 or 500 M glucose. The lines are exponential fits (*R*2 >0.924, >0.985 and >0.992, respectively).

The values of 1/ (mean + SD, n = 1200 over 2 min) as function of [-glucose] are shown in Fig. 4. The line is linear fit and the value of *k*q (101.1 sec-1 M-1) is the negatives of the slope. The value of 1/for air-saturated solution (without glucose) was 28,330 sec-1 (coefficient of variation, Cv = 12%), for [-glucose] = 125 M 5,650 sec-1, and for O2-depleted solution (with 500 M glucose, 1/ 2,875 sec-1 (Cv = 1%). The high values of Cv for the airsaturated solutions were due to the lower phosphorescence intensities with high [O2] (little light reaching the photomultiplier tube). The corresponding lifetimes () were 52 sec, 177 sec and 352 sec, respectively. Oxygen concentration was calculated using, *k*q[O2].

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 243

with lower perforin (a cytolytic protein produced by natural killer lymphocytes) expression on CD8+ T-lymphocytes (Jiang et al., 2008). A dose-related decrease in DNA synthesis in lymphocyte cultures (with and without mitogens) is found in mice exposed *in vivo* to aflatoxin B1 (Reddy et al., 1987). A decrease in DNA synthesis is also observed in normal splenic mouse lymphocytes cultured *in vitro* with >10 M aflatoxin B1; a decrease in RNA synthesis is observed at dosing >25 M and a decrease in protein synthesis at dosing >100

The phosphorescence oxygen analyzer is used to monitor the effects of aflatoxin B1 on human lymphocyte mitochondrial oxygen consumption. These experiments investigate whether aflatoxin B1 impairs respiration of the lymphoid tissue, an organ that is typically targeted by this potent mycotoxin. Aflatoxin B1 (1.0 mg = 3.2 micromol) was freshly dissolved in 1.0 mL dry methanol and immediately added to cell suspensions with vigorous mixing. Alternatively, aflatoxin B1 powder was directly added to the cell suspension with vigorous mixing. The concentrations were determined by the absorbance at 350 nm (10 L aflatoxin B1 stock solution or cell-free supernatant in 1.0 mL dry methanol), using an extinction coefficient of 21,500 M-1 cm-1 (Nesheim et al., 1999); the aflatoxin B1 excitation wavelength is 366 nm and the emission wavelength 455 nm. The reactions were carried out

PBMC (0.6 x 107 cells/mL) were suspended in 6.0 mL PBS, 10 mM glucose, 3 M Pd phosphor and 0.5% fat-free bovine serum albumin. The mixture was divided into 2 equal aliquots. Methanol (25 L per mL, Fig. 5, left panel) or aflatoxin B1 (25 M, Fig. 5, right panel) was then added and the incubation continued at 37oC (open to air with gentle stirring). At *t* = 10 and 110 min, 1.0 mL of each mixture was simultaneously placed in the instruments for O2 measurement. The rate of respiration (*k*, in M O2 min-1) for *t* = 10 to 78 min for the methanol-treated cells was 2.0 and for the aflatoxin B1-treated cells 2.1. The values of *k* for *t* = 110 to 174 were 2.2 and 1.2, respectively (corresponding to 45% inhibition

PBMC were incubated at 37oC with 25 μL per mL methanol (left panel) or 25 μM aflatoxin B1 (right panel). Minute zero corresponds to the addition of aflatoxin B1. At t = 10 and t = 110 min, 1.0 mL of each mixture was simultaneously placed in the instruments for O2 measurement. Rates of respiration (k) were calculated from the best-fit linear curves. Additions of 5.0 mM NaCN and 50 g/mL glucose

Fig. 5. Effect of aflatoxin B1 on human PBMC respiration.

M (Reddy et al., 1989).

in glass vials and protected from light.

of lymphocyte respiration).

oxidase are shown.

The reaction mixtures contained PBS, 3 M Pd phosphor, 0.5% fat-free bovine serum albumin, 50 g/mL glucose oxidase and shown concentrations of β-glucose. The values of 1/τ (mean ± SD, n = 1200 flashes over 2 min) as a function of [β-glucose] are shown. The lines are linear fits.

Fig. 4. Calibration with -glucose plus glucose oxidase
