Oxygen Consumption at Constant Oxygen Concentration



Respirometry



Previously, the oxygen consumption rate (OCR or VO2) of cells is measured using respirometry.
Cells are placed in a sealed chamber, usually stirred, and an oxygen electrode measures the oxygen concentration of the suspending medium.

No oxygen can enter or leave the chamber so that the oxygen concentration of the medium decreases as the cells consume oxygen. The rate of change of the oxygen concentration (dO2/dt) is equal to minus the oxygen consumption (VO2), e.g.



The oxygen consumption is measured from the rate that the oxygen concentration decreases.

Respirometry is an extremely powerful technique but has several disadvantages:
  • The oxygen concentration is continuously changing
  • There is limited time before the oxygen is completely consumed and the cells become anoxic
  • Experiments usually begin with high concentrations of oxygen which are non-physiologica


A typical study using respirometry to measure oxygen consumption after addition of oligomycin (O) to inhibit oxygen consumption and uncoupler (U) to stimulate oxygen consumption.

Iberius



The Iberius uses a different technique. A loop of fine-gauge silicone tubing is placed in the cell suspension and a nitrogen/oxygen gas mixture is passed through the tubing. The tubing is highly permeable to gases and this delivers oxygen to the cell suspension.

In this case, the rate of change of the oxygen concentration (dO2/dt) is equal to minus the oxygen consumption (VO2) plus the delivery of oxygen (DO2) by the tubing, e.g.



Now, when the oxygen delivery equals the oxygen consumption, then the oxygen concentration is constant. Furthermore, when the oxygen concentration is constant, the oxygen consumption is just equal to the oxygen delivery.

The Iberius uses a computer-controller gas blender to set the oxygen fraction in the tubing, and has a feedback loop to set the oxygen at the desired concentration. It also mixes carbon dioxide to maintain the pH balance.

The Iberius has the following advantages over respirometry:
  • The oxygen concentration is constant during the study
  • There is no time limit on a study
  • The oxygen concentration can be set anywhere from anoxia to hyperoxia
  • The oxygen can be reversibly reduced to zero during a study to fully reduce the hemes of the ETC and the mitochondrial NADH pool.

Attempts have been made to maintain constant oxygen concentration by unsealing the chamber and blowing oxygen over the surface of the cell suspension. This has the following disadvantages

  • It generates oxygen gradients from top to bottom of the chamber as the stirrer has poor up-down mixing.
  • The chamber is not sealed and so oxygen leaks in and the oxygenation cannot be completely zero.
  • Oxygen consumption cannot be calculated if an unknown quantity of oxygen is leaking into the chamber.

A typical study using the Iberius to measure oxygen consumption after addition of oligomycin (O) to inhibit oxygen consumption and uncoupler (U) to stimulate oxygen consumption.


Oxygen Electrodes and Oxygen Optodes



Typically, respirometry instruments use oxygen electrodes to measure the oxygen concentration in the cell suspension. These electrodes need to be calibrated every day and their calibration can drift during the day.

The Iberius system uses an oxygen optode which measures oxygen concentration using a phosphorescent compound which is quenched in the presence of oxygen. The Iberius measures the lifetime of the phosphorescence, which is extremely stable. These optodes maintain their calibration week in, week out.
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