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There are a number of methods to measure hypoxia in tissues; all have advantages and disadvantages. Three key methods that are relevant to the discussions in this paper are below (for reviews see Refs 142,143,144,145,146,147).

Hypoxia marker drugs

These drugs are 2-nitroimidazoles; after entering cells they undergo 1-2 electron reduction in cells and the reduced drug is a highly reactive free radical that binds to macromolecules, including proteins148. However, when oxygen is present, the drug is oxidized and reverts back to its original state, allowing it to diffuse out of the cell and eventually into the circulation. The rate of protein binding of the reduced drug increases exponentially with a decrease in partial pressure of O2, particularly below 10 mmHg144, 149. The presence of drug-protein adducts can be detected immunohistochemically using antibodies specific for the drug protein adduct. 18F-labelled versions of these drugs are also being developed for positron-emission tomography imaging150, 151.

Hypoxia marker proteins

As HIF1 upregulates the synthesis of many proteins, it has been proposed that identification of such proteins in tissues could be markers of hypoxia. Literally dozens of such proteins have been studied at the preclinical level and in clinical trials152. Some of the more promising endogenous markers include carbonic anhydrase IX (CA9), plasminogen activator inhibitor 1 (PAI1, also known as SERPINE), osteopontin and lysyl oxidase. Combinations of markers might prove to be better predictors of clinical outcome than any single marker153.

Oxygen electrodes and optical probes

Oxygen can be measured in any aqueous media using polarography. In principle, two electrodes are placed into the medium and a polarizing voltage of - 0.7 volts is applied across them. This voltage corresponds to the binding energy of outer shell electrons of oxygen. The electrons are captured by the cathode and the current generated is linearly proportional to oxygen concentration. In practice, the cathode is embedded into a needle that can be introduced into tissues and the anode is placed on the body surface. This technique has been used extensively in preclinical and clinical studies; the presence of hypoxia is an independent predictive factor for poor prognosis in many different tumour types154. Optical probes have also been developed: these are implanted into tissues and contain a fluorochrome that emits fluorescent light with a certain decay rate when illuminated. The rate of fluorescent light decay is proportional to the oxygen concentration in the region of measurement. These probes yield data similar to that of the oxygen electrode6.

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