ABSTRACT
Like the majority of lower vertebrates, the newt Triturus carnifex holds varying quantities of melanin and hemosiderin in the Kupffer cells of the liver. Following hypoxic treatment, the amount of these two pigments can increase to such an extent that they can occupy nearly a quarter of the surface of histological sections. A group of six specimens, anesthetised with chlorbutol, were subjected to hypoxic treatment by keeping them in a respiratory chamber containing degassed water under vacuum, with only 1.1 ppm of residual oxygen, until they had consumed the oxygen completely (4 hours, at a temperature of 18 degrees C). Using hematological and histochemical techniques and computerised image analysis, it has been shown that hypoxic animals not only increase the extent of the melanic areas of the liver from about 5-7% to almost 24% compared to control groups kept under two different respiratory conditions (6 anesthetised specimens exposed to the air and 6 submerged in normoxic water), they also went through a remarkable hemolytic process to justify a parallel increase in hemosiderin deposits. Melanin was extracted from the liver by keeping fragments of the organ for one hour at 37 degrees C in an oxidising solution (20 mL of benzyl alcohol, 10 mL of acetone, 5 mL of 10% hydrogen peroxide, and 4 drops of concentrated ammonia solution), then quickly rinsing them in 50% acetone and subsequently letting them stand for 6 hours in 10 mL of distilled water alkalised to pH 12 with a drop of ammonia solution. The extract was then left to sediment at pH 2.5 and the black precipitate washed and dried under vacuum. Elemental and spectrophotometric analyses revealed a significant presence of purines in the melanic pigment. This phenomenon can be explained by the animals' need under hypoxic crisis to rapidly neutralise purines resulting from lysis of the nucleated red blood cells by introducing them into an inert molecular complex. A partial model of structure is proposed here. Synthesis of the mixed polymer is possible through the well-known capacity of ferrous iron to activate tyrosinase (the enzyme responsible for melanogenesis) even in the absence of DOPA.
