Title: Antioxidant Activity In Human Plasma.
Key words: Epidemiological, oxidative stress, antioxidants, free radicals, peroxidation, total antioxidant assays
Date: February, 2000
Category: 14. Measurement
Author: Dr van Rhijn
Epidemiological studies confirm that causes of death over the last century are increasingly related to oxidative stress, and that the population at large is at risk of antioxidant (preventative and radical-scavenging) deficiency. The subsequent interest in antioxidant assays produced several available methods, the majority being inhibition assays of the free radical generation, using quantifiable1 fluorescent or chemiluminescent end points, which can be inhibited and measured by the addition of an antioxidant.
Oxidative Stress states
Oxidative stress refers to the situation of a serious imbalance between production of reactive oxygen species (ROS) / reactive nitrogen species (RNS) and antioxidant defence2. Increased generation of ROS/RNS in vivo can lead to the depletion of one or more antioxidants, which can be measured as an index of oxidative stress3. However, this does not necessarily imply oxidative damage or cell injury. The relative importance of antioxidants4 depends upon which, how, when and where ROS/RNS are generated, and which target of damage is measured.
Abnormally low levels of TAA are associated with numerous oxidative stress-related clinical conditions such as ischaemia (cerebral & cardiac5), atherosclerosis, diabetes mellitus, pulmonary disease, septic shock, inflammations and premature neonates. The ability to measure TAA may be clinically relevant for clinical diagnosis and monitoring of nutritional supplementation.
Methods for Total Antioxidant Activity Measurements
Due to the complexity of the antioxidant defence system, oxidative stress is measured as total antioxidant activity (TAA) rather than attempting to measure each individual component involved. Several assays have been developed, such as the TRAP 6 (total [peroxyl] radical trapping antioxidant parameter). This measures peroxidation of lipids in serum on exposure to azo initiators. RO2 react and deplete plasma antioxidants prior to attacking the lipids 7 to cause peroxidation, and this lag period can be measured and calibrated with a known antioxidant (the agreed standard: Trolox – a water-soluble vitamin E analogue). The oxygen radical absorbance capacity ORAC 8 is an assay variant using phycoerythrin (red algae photosensitive protein) to quantify antioxidant capacity. The ABTS 9 is an automated spectrophotometric technique measuring oxidation of ABTS to the highly coloured radical cation ABTS· + by metmyoglobin/H2O2, horseradish peroxidase/H2O2 or by adding MnO2. Antioxidants decolorise the radical, which is recorded. The FRAP method measures the ability of plasma antioxidants (Vit E, ascorbate, urate but not albumin) to reduce Fe3+ to Fe2+ at low pH.
Albumin and uric acid account for around 57% of the total antioxidant activity in plasma (measured TRAP values) and the residual activity (calculated TRAP value) is referred to as the ‘antioxidant gap’10. This reflects the combined activity of other plasma antioxidants (ascorbic acid, a -tocoferol, b -carotene etc). The trolox equivalent antioxidant capacity11 (TEAC) can be used to compare the antioxidant activity of different compounds and to explore the antioxidant content of complex mixtures, which may be more appropriate. Each method provides different reference intervals, but there is a correlation between TAA and the TRAP.
Total antioxidant assays of plasma provide a global picture of relative antioxidant activities during clinical conditions. Results can be misleading due to the variability of albumin or uric acid in certain diseases, and should be interpreted in light of the assay used. Also, their ‘relative importance’ is not necessarily a reflection of their concentration in extra-cellular fluid.