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EARLY investigations (ROBERTS, LOWE, GUTH and JELINEK, 1958) of the ninhydrin positive substances of the whole brain of rats indicated that these substances were unchanged by various drug treatments or physiological stresses. It was mentioned at that time that studies of the whole brain could be misleading. Later work with whole brains of rats and mice ( HAKKINEN and KULONEN, 1961 ; SOEP and JANSSEN, 1961;DEROPP and SNEDEKER, 1961;KAMRIN and KAMRIN, 1961) revealed that the administration of different drugs does alter the concentration of several of the free amino acids. Although no meaningful pattern of changes was evident, these reports raised the possibility that a meaningful pattern of changes could be found through a more detailed study with more refined techniques.In recent years the development of dual column (SPACKMAN, STEIN and MOORE, 1958) and single column (PIEI. and MORRIS, 1960) amino acid analysers has facilitated the relatively rapid separation and determination of ninhydrin positive substances in biological extracts. The studies of TALLAN, MOORE and STEIN (1954) had already established the large number of ninhydrin positive substances present in the brain of a cat as revealed by column chromatography, including unknown peaks. This paper describes in detail the procedures used in establishing the concentrations of ninhydrin positive substances in four areas of the rat brain. Other body organs and tissues were analysed for comparison to establish the presence of unknown substances peculiar to the brain. M E T H O D S Preparation of brain extracts.Male albino rats from the Suffield Experimental Station animal colony, weighing 225-250 g and starved 16-24 hr were used in all experiments. After decapitation the brains were quickly removed, subdivided, weighed, homogenized in 10ml 95% ethanol and acidified with 1 % ~N -H C~. A Potter-Elvehjem homogenizer with a teflon pestle was used. The brain was subdivided into four areas: the pons medulla, cerebellum, hemispheres free of the basal ganglia and olfactory lobes, and the remainder, which was designated as midbrain. Approximately 3 min passed between decapitation and immersion of the brain areas into the acidified ethanol. The hemispheres were always weighed and homogenized first.The homogenized fractions were placed on a rotary agitator at room temperature for 30 min to improve the extraction. After centrifuging and decanting the supernatant, the residue was re-treated once with acidified ethanol and once with 95% ethanol. The combined supernatants were evaporated to dryness under vacuum at room temperature on a rotary evaporator. The final residue was treated once with 2 ml distilled water for 30 min. The suspension was centrifuged for 10 min at 30,000 g and the final supernatant was immediately stored at -15". Samples were normally stored for 24 to Abbreviation used: AGPA, 2-amino-3-guanidinopropionic acid.

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Several high pressure liquid chromatographic methods for the separation of conjugated and free bile acids are presented. A mixture of synthetic conjugated bile acids has been separated by reverse-phase systems consisting of either a Waters Associated' "fatty-acid analysis" or a muBondapak/C18 column eluted with a mixture of 2-propanol/potassium phosphage buffer (pH 2.5 or 7.0). The major conjugated bile acids present in the gallbladder bile of obese subjects have been analyzed each in less than 30 min and quantitated with a U.V. detector set at 193 nm. Some of the 5 alpha- and 5 beta-isomers of conjugated bile salts could be resolved in straight-phase systems on Corasil II or muPorasil columns. Mass spectra of the conjugated bile acids obtained by electron impact were characteristics of the type of amino acid attached to the side chain, and the number of hydroxyl substituents on the nucleus. Most of the isomers could readily be differentiated by the relative intensities of the fragment ions.

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