The heme degradation pathway converts heme to bilirubin (BR), carbon monoxide and ferric
ion. The two NADPH dependent enzymes which are responsible for the conversion are heme
oxygenase (HO), which converts heme to biliverdin (BV), and biliverdin reductase (BVR),
which converts BV to BR. HO forms the rate limiting step of the degradation and plays an
important role for oxidative stress an cell homeostasis. BVR plays also an important role in
pathologic conditions involving biases of the redox balance. Therefore it is frequently
necessary to determine changes of the activity of the heme degradation pathway. The
biochemical enzymatic assay used previously is based on the original coupled enzymatic
assay described by Tenhunen et al. (1968) subsequently followed by an extraction of the
formed BR with an organic solvent and a subsequently spectrophotometric measurement to
determine the concentration. This assay works well for tissues with high enzymatic activity
but for tissues with low activity or small sample volumes the formed amount of BR is not
sufficient to allow precise determination of BR concentration and may fall below the limit of
detection for the spectrophotometric measurement. Therefore, the aim of this study was to
optimize the HO assay for small sample volumes. During the optimization we had to solve
several problems: First the high protein content of the samples interfered with the
extractability of BR, second the original extracting solvent chloroform was not viable for very
low BR concentrations. We solved the protein problem by adding caffeine solution prior to
the extraction, which almost negated the protein effect and enhanced the BR yield by factor 2.
The problem of the extracting solvent was solved by substituting chloroform with benzene.
Doing that allowed us to halve the overall assay volume, which further increased the
sensitivity. By comparing the classic protocol with the optimized protocol we have shown that
the yield of the assay normalized for protein is enhanced by a factor from 3,5 to 4,6 depending
on the enzymatic activity of the investigated tissue.