Investigation of antioxidant capacity parameters

Direct measurement is difficult due to the short half-life of free radicals (VR). Substances formed by their action are determined.

Direct Determination

Determination of oxygen radicals

  • pulse radiolysis – radicals are generated by ionizing radiation
  • electron spin resonance spectrometry (ESR) – identification based on spin changes
  • chemiluminescence method

Determination of nitrogen radicals and their adducts

  • nitric oxide – it is very difficult to determine
  • methods as in oxygen
  • most often indirect methods - nitrites, nitrates or substances modified by nitration - nitrosohemoglobin

Determination of radical generating substances

  • xanthine oxidase – produces superoxide
  • determination of transition metals – Fe, Cu (they catalyze reactions where free radicals are formed)

Indirect measurements

  • most often determination of lipoperoxidation products, adducts with DNA

VR damage products

NK damage

  • the most significant (irreversible) damage – by the hydroxyl radical
  • main product – thymine glycol and 8-hydroxyguanine
  • repair enzymes remove them from the cells - we can determine them in the urine
8-hydroxyguanine
Thyminglycol

Damage to proteins and AMK

  • many damage mechanisms, little used
  • sensitive method is for measuring carbonyl residues from lysine.

Lipoperoxidation

  • in direct connection with the formation of free radicals
  • most common – determination of malondialdehyde (MDA) – reaction with thiobarbiturate forms a colored complex, non-specific, also reacts with e.g. bilirubin, DNA
  • also other aldehydes (e.g. 4-hydroxynonenal)
  • conjugated dienes – characteristic UV absorption (234 nm)
  • measurement of hydrocarbons in exhaled air
  • isoprostanes - by peroxidation of products arachidonic acids

Oxidized LDL

  1. change of the delay phase in stimulated peroxidation - examines the ability of LDL to cope with oxidative stress
  2. determination of oxLDL – extraction, oxFFA is determined at 234 nm

Antioxidant protection of the organism

Total antioxidant capacity

  • artificial formation of free radicals in biological material - we measure the ability to slow down or stop this reaction
  • TRAP determination – plasma capacity after adding the generator – conversion to Trolox capacity – 1 trolox molecule has 2.0 units when using TRAP – disadvantage – end-point oxygen electrode
  • more commonly used ABTS – inhibition of the ABTS radical cation

Antioxidant enzymes

  • determination of SOD rather indirectly, determination of catalase – rarely

Antioxidant substrates

  • vitamins A, E, C
  • determination of thiols is irrelevant (they are on albumin, they have a long half-life)
  • also others - ubiquinone Q, lipoate, flavonoids..., using high-performance (high-pressure) liquid chromatography (HPLC)

References

  • SCHNEIDERKA, Peter. Chapters in Clinical Biochemistry. 2. edition. Prague : Karolinum, 2004. ISBN 80-246-0678-X.