**4. Principles and tools of the oxygen measurement**

O2 concentration is determined from the phosphorescence decay rate (1/) of the *palladium (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin* (Pd phosphor). This measurement is based on quenching the phosphorescence of Pd phosphor by O2 (Lo et al., 1996). The probe has an absorption maximum at 625 nm and an emission maximum at 800 nm. Samples are exposed to light flashes (10 per sec) from a pulsed light-emitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc), Wheeling, IL. Emitted phosphorescent light is detected by a Hamamatsu photomultiplier tube (PMT #928) after first passing it through a wide-band interference filter centered at 800 nm. Amplified phosphorescence is digitized at 1-2 MHz using an analog/digital converter (PCI-DAS 4020/12 I/O Board) with 1 to 20 MHz outputs (Computer Boards, Inc.). Pulses are captured using a developed software program at 0.1 to 4.0 MHz, depending on speed of the computer (Souid, 2003).

The values of 1/ are linear with dissolved O2 concentration: 1*k*q[O2], where 1/ = the phosphorescence decay rate in the presence of O2, 1/o = the phosphorescence decay rate in the absence of O2, and *k*q = the second-order O2 quenching rate constant in sec-1 M-1 (Lo et al., 1996).

Cellular respiration is measured at 37oC in 1-mL sealed vials. Mixing is carried out with the aid of parylene-coated stirring bars. The respiratory substrates are the endogenous metabolic fuels supplemented with glucose. In cell suspensions sealed from air, [O2] decreased linearly with time, indicating the kinetics of cellular mitochondrial O2 consumption is zero-order. The rate of respiration (*k*, in M O2 min-1) is thus the negative of the slope d [O2]/d*t*. Cyanide markedly inhibited respiration, confirming O2 is consumed mainly by the mitochondrial respiratory chain.

## **5. Developed software program and instrument description**

The software program was developed using Microsoft Visual Basic 6 (VB6) programming language, Microsoft Access Database 2007 (Access) database management system, and Universal Library components developed by the electronic board company, Measurement Computing, for use with Microsoft Visual Basic 6 programming language

electrochemical potential Δψ (Ricci et al., 2004). Thus, induction of apoptosis is directly

Dissolved O2 is expressed in mm Hg, mL O2 per L, mg O2 per L, or mol per L (M). For conversion, a partial pressure of O2 (*P*O2) of 1.0 mm Hg = 0.03 mL O2 per L; 1.0 mL O2 per L = 1.4276 mg O2 per L; and 1.0 mg O2 per L = 1000/32 M. In *freshwater* at 760 mm Hg and 20°C, dissolved [O2] is 9.1 mg/L, or 284 M. Using a Clark electrode, *P*O2 of the reaction mixture phosphate-buffer saline (PBS), 10 mM glucose and 0.5% fat-free bovine serum albumin is 170.5 + 6.6 mm Hg (n = 4), or 228 + 9 M. The 56 mm Hg difference between [O2] in freshwater and the reaction solution reflects the effect of salinity on dissolved O2 (Weiss,

O2 concentration is determined from the phosphorescence decay rate (1/) of the *palladium (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin* (Pd phosphor). This measurement is based on quenching the phosphorescence of Pd phosphor by O2 (Lo et al., 1996). The probe has an absorption maximum at 625 nm and an emission maximum at 800 nm. Samples are exposed to light flashes (10 per sec) from a pulsed light-emitting diode array with peak output at 625 nm (OTL630A-5-10-66-E, Opto Technology, Inc), Wheeling, IL. Emitted phosphorescent light is detected by a Hamamatsu photomultiplier tube (PMT #928) after first passing it through a wide-band interference filter centered at 800 nm. Amplified phosphorescence is digitized at 1-2 MHz using an analog/digital converter (PCI-DAS 4020/12 I/O Board) with 1 to 20 MHz outputs (Computer Boards, Inc.). Pulses are captured using a developed software program at 0.1 to 4.0 MHz, depending on speed of the

The values of 1/ are linear with dissolved O2 concentration: 1*k*q[O2], where 1/ = the phosphorescence decay rate in the presence of O2, 1/o = the phosphorescence decay rate in the absence of O2, and *k*q = the second-order O2 quenching rate constant in sec-1 M-1

Cellular respiration is measured at 37oC in 1-mL sealed vials. Mixing is carried out with the aid of parylene-coated stirring bars. The respiratory substrates are the endogenous metabolic fuels supplemented with glucose. In cell suspensions sealed from air, [O2] decreased linearly with time, indicating the kinetics of cellular mitochondrial O2 consumption is zero-order. The rate of respiration (*k*, in M O2 min-1) is thus the negative of the slope d [O2]/d*t*. Cyanide markedly inhibited respiration, confirming O2 is consumed

The software program was developed using Microsoft Visual Basic 6 (VB6) programming language, Microsoft Access Database 2007 (Access) database management system, and Universal Library components developed by the electronic board company, Measurement Computing, for use with Microsoft Visual Basic 6 programming language

linked to mitochondrial dysfunction (Green and Kroemer, 2004).

**4. Principles and tools of the oxygen measurement** 

**3. Expressions of dissolved oxygen** 

1970).

computer (Souid, 2003).

mainly by the mitochondrial respiratory chain.

**5. Developed software program and instrument description** 

(Lo et al., 1996).

(http://www.mccdaq.com/daq-software/universal-library.aspx). It allows direct reading from the PCI-DAS 4020/12 I/O Board (http://www.mccdaq.com/pci-dataacquisition/PCI-DAS4020-12.aspx). The software utilizes a relational database that stores experiments, pulses and pulse metadata, including slopes. Pulse identification is performed by detecting 10 phosphorescence intensities above 1.0 volt (by default). Peak identification is performed by the program which detects the highest 10% data points of a pulse and chooses the point in the group that is closest to the pulse's decay curve. Depending on the sample rate, a minimum number of data points per pulse is set and used as a cutoff to remove invalid pulses with too few data points (Shaban, 2010).

Main advantages of the developed program over commercially available packages (e.g., DASYLab™ or TracerDAQ™) are provision of full control and customization of the data acquisition, storage and analysis. The choices of VB6 and Access as programming and storage environments are due to their availability, simplicity, widespread use and VB6 components that read directly from the PCI card made available by Measurement Computing. Table 1 displays identified tasks of the program. Fig. 1 shows a picture of the data acquisition system and the developed software program. Fig. 2 shows a reaction vial.

Experiment identification (title, date, time and sample rate)

Reading directly from the PCI card at the fastest possible rate

Distinguishing pulse data from non-pulse data

Allowing a fuzzy detection of the pulse peak

Calculating the exponential decay rate (1/) and lifetime () of each pulse

Storing each pulse data points, along with the peak, decay and lifetime values

Viewing a representative pulse every 10 sec

Viewing decay rates (1/) in a second graph

Ability to pause and place a marker with a note

Ability to remove erroneous (incomplete) pulses and adjust peak values if necessary

Ability to copy pulse or slope data to clipboard for further analysis

Ability to access a previous experiment, review a pulse with its metadata, markers and associated notes

Table 1. Identified tasks of the customized software program for data acquisition, storage and analysis

Phosphorescence Oxygen Analyzer as a Measuring Tool for Cellular Bioenergetics 241


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,

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

**6. Instrument calibration** 

serum albumin are shown in Fig. 3.

*k*q[O2].

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

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

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