Neonatal
hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of
oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment. Decreased
adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy
phosphate bonds are degraded. This leads to increased levels of
adenosine which is subsequently degraded to
inosine,
hypoxanthine,
xanthine, and finally to
uric acid. The final two steps in this degradation process are performed by
xanthine oxidoreductase. This
enzyme exists in the form of
xanthine dehydrogenase under normoxic conditions but is converted to
xanthine oxidase (XO) under
hypoxia-reperfusion circumstances. Unlike
xanthine dehydrogenase, XO generates
hydrogen peroxide as a byproduct of
purine degradation. This
hydrogen peroxide in combination with other
reactive oxygen species (ROS) produced during
hypoxia, oxidizes
uric acid to form
allantoin and reacts with
lipid membranes to generate
malondialdehyde (MDA). Most mammals, humans exempted, possess the
enzyme uricase, which converts
uric acid to
allantoin. In humans, however,
allantoin can only be formed by ROS-mediated oxidation of
uric acid. Because of this,
allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have
uricase. We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure
biochemical markers of neonatal
hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for
uricase-generated
allantoin.
Purine metabolites were linked to
hypoxia as early as 1963 and the reliability of
hypoxanthine,
xanthine, and
uric acid as biochemical indicators of neonatal
hypoxia was validated by several investigators. The HPLC method used for the quantification of
purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of
allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of
MMDA were described elsewhere. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al.
Xanthine oxidase activity was measured by HPLC by quantifying the conversion of
pterin to
isoxanthopterin. This approach proved to be sufficiently sensitive and reproducible.