DEMONSTRATION OF SOME COMPOUNDS PRESENT IN THE PINEAL ORGAN OF THE ALBINO RAT BY HISTOCHEMICAL METHODS AND PAPER CHROMATOGRAPHY

Abstract

In histological sections of the rat pineal organ stained with the osmic acid-sodium iodide technique of Champy, many pinealocytes show black-stained drop-like or granular inclusions of a variable number and size. Their number is often so large that parts of cells or entire cells are evenly blackened. The chemical identification of these inclusions has been the object of the present investigation. Because, in vitro, every osmiophilic compound is also stained with the mixture of Champy it has been assumed that cellular substances will stain with this mixture if reacting with osmic acid, the component of major importance of Champy''s fluid. In sections the presence of the following osmiophilic compounds has been tested witn more or less specific cytochemical staining techniques: lipids, aromatic amines (catecholamines), ascorbic acid and indoles. Rather surprisingly, lipids and ascorbic acid only could be demonstrated in this way. Relatively large lipid droplets were shown. A few of the smaller inclusions visualized with the technique of Champy may consist of ascorbic acid. The majority, however, contain a still unidentified compound(s), probably of a non-lipid nature. The identity of this material with carbohydrates, nucleic acids or histamine can be excluded, these latter substances being non-osmiophilic. Amino-acids, staining with ninhydrin in chromatograms of pineal extracts, did not give a positive spot test with Champy''s fluid. Pineal indoles - serotonin, melatonin, 5-methoxyindole-3-acetic acid and 5-hydroxy-indole-3-acetic acid - could not be demonstrated with any of the cytochemical staining techniques used. However, serotonin was shown to be present in the epiphysis by paper chromatographic analysis and melatonin by chromatography followed by bioassays applied to larvae of Xenopus laevis. It is improbable that the black inclusions in the pinealocytes stained with the Champy-technique contain any of the pineal indoles because it was shown that the cytochemical methods, specific for indoles, gave negative results in the sections although being more sensitive than the fluid of Champy. These negative results may be caused by the extremely small quantities present, especially if the total indole content of an epiphysis was diffusely distributed throughout the pineal tissue. Possibly, however, staining is prevented by the indoles being masked by kinkage to other compounds. The total amount of melatonin present in a single rat epiphysis has been determined by means of bio-assays, comparing the pigment granule concentrating effect in melanophore s of Xenopus larvae caused by the aqueous extract of a single pineal organ with the similar effect of several known concentrations of pure synthetized melatonin dissolved in water. This quantity is 5 x 10-4 gamma. Comparison with data from the literature on the bovine epiphysis showed that the total content of melatonin of a single epiphysis in cattle is 0.04 gamma. Furthermore, it was calculated that the amount of pineal melatonin per unit weight is two times larger in the rat than it is in cattle, the figures being approximately 0.4 gamma/g and 0.2 gamma/g respectively.