Document ZJgOj7rrBjz6p3mOrjp9kxqyZ

~V -! 10/25/57 C* Stevens Problems What happens to tetraethyllead (TEL) in the body ? Work of previous investigators; No publication bearing directly upon this problem has appeared during the past year. Earlier work is cited in my previous reports. Plan of attack: Two main objectives were selected for investigation: (1) reactions of TEL in vivo, - their rates, products and mechanisms, and (2) distribution of TEL and of its lead-containing decomposition products among tissues. To approach these objectives requires the development of analytical methods that will be specific for TEL and for its decomposition products. Progress, 1955-56: The work during these years was concerned primarily with devising analytical methods for detection of TEL and of triethyllead ion in tissues. Fairly conclusive evidence was obtained of the presence in liver of TEL after its inhalation. This was published. Development of methods for the triethyllead ion then proceeded, first by synthesis of various triethyllead salts and then by studies of their behavior in systems of interest, e.g., their recovery from liver homogenates, their partitioning between immiscible solvents, their solubility/ 0003450 N6621 10/ 25/57 A - " - 2 - C. Stevens and so on. This led to the demonstration of triethyllead ion in extracts of livers removed from animals one day after they had inhaled TEL. The evidence for this was mainly infrared spectra and lead analyses, it remains a little uncertain whether the triethyllead ion was actually in the livers or might have been produced from TEL and diethyllead ion during the isolation procedure. In this way the diethyllead ion was drawn into the work, that something could be learned of its stability and possible occurrence in tissue. Progress, 1957: Search for the diethyllead ion in tissue has been our main concern this year. As with the triethyllead ion, synthesis of salts of the diethyllead ion was necessary. These salts were tested as they were made for properties that might be exploited in seeking for diethyllead ion. The dichloride and dibenzoate have been the most useful derivatives. Scant indication of the presence of diethyllead ion had been obtained in our earlier work on tissue. A reason for this soon became evident. The diethyllead ion is much more firmly bound to tissue than is the triethyllead ion. For example, with, benzene as the solvent, efficiency of extraction of diethyllead dichloride from liver is only one-tenth that of triethyllead chloride. This firmer bond may be reflected in a more deleterious effect in vivo, but the only report known to me concerning the toxicity of diethyllead salts is that of Saunders and Stacey (1949) who observed no sternutatory action from the dichloride; the point remains uninvestigated. 0003451 10/25/57 -3 C. Stevens To displace the diethyllead ion from its bonds with tissue a variety of methods have been used, many of them with little success (as noted in my weekly reports). The two best methods developed thus far have as their initial steps (a) the formation of benzoates and their extraction with methylene chloride and (b) the formation of dithizonates and their extraction with chloroform. Each of these methods has its own serious disadvantages; each gives a recovery in the initial steps of about 50/ of the added diethyllead dichloride. Application of method (a) to livers taken from rats a day after inhalation of TEL indicates that diethyllead ion is probably present at that time. More work upon intoxicated animals has been foregone until the procedure of isolation can be further developed. This is the work now in hand. An observation with therapeutic implications was made in the course of the analytical work. The diethyllead ion was found to form complexes in water with Versene (ethylenediaminetetraacetic acid) and with beta-mercapto-ethylamine as shown by ultraviolet spectroscopy. These complexes remained in the aqueous phase when shaken with iso-cctane. Because of this unpromising behavior the complexes were investigated no further; their stability in the presence of tissue remains unknown. Another thiol, o-aminobenzenethiol, gave no spectroscopic evidence of complex formation in water although it is known to react with inorganic lead in 50/ dioxane (Freiser et al.,1952). ft g 0003452 10/25/57 -4- C. Steven3 Several experiments designed to discover whether TEL acts directly upon muscle cells or acts through the nervous system or both were performed on rats by Arthur Asbury with our collaboration. No summation of response in merve-muscle preparations was found within a few hours after beginning inhalation of TEL, indicating that no inhibition of choline - esterase at the myoneural junction had occurred by then. Whether such inhibition occurs after tremor and other signs of intoxication are evident was not determined because of technical difficulties. Proposal for continuation: I suggest work be continued with these objectives: 1. Completion of evidence for occurrence of both triethyllead and diethyllead ions in liver after inhalation of TEL. 2. Development of methods for measurement of TEL and of each of its organolead derivatives in tissue. 5. Application of the quantitative technics so developed to the problems noted above under "Plan of attack". 0003453