Studies on the Chemistry of Wax D of BCG

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Abstract
“Purified wax”, as isolated by Anderson (1), was a complex lipide prepared from acid fast bacteria by extraction with chloroform. Some of its hydrolytic products were chemically identified by him and his collaborators. Recently, Asselineau (2) fractionated “purified wax” into Wax C and Wax D. Wax C was a mixture of high molecular compounds of low melting points, which contained mycolic acids and carbohydrates but no nitrogeneous compounds as constituents, while Wax D was reported to consist mainly of high molecular lipopolysaccharides of higher melting points containing small amounts of phosphorus and nitrogen. Following the first report that the toxic factor of tubercle bacilli known as “cord factor” was concentrated in Wax C fraction (3), chemical investigations of the constituents in that fraction were performed intensively, chiefly by the use of column chromatography, with the results that cord factor and some other complex lipides were isolated in pure forms and their characteristics were clarified (4). Wax D, on the other hand, has not been investigated as fully as Wax C (5). Wax D, as well as similar preparations, seems to playa biologically significant role in manifesting toxicity of tubercle bacilli (6), in causing tuberculin allergy as an adjuvant (7), and in other related phenomena (8). However, there are many conflicting views (89) on these biological activities. Such contradictory views indicate the need for better methods for resolution of complex lipides in the fractions and chemical characterization of the individual component. It was previously shown by the present author that Wax D of BCG usually contained 3 percent of inositol (10). The present paper describes a method for fractionating Wax D of BCG into two components by silicic acid column chromatography. One fraction was a lipide mixture of low melting point, lacking phosphorus and nitrogen, from which “cord factor” (trehalose-6,6'-dimycolate) was isolated. Another fraction consisted of a mixture of lipides of relatively high melting point containing phosphorus and inositol. From the latter fraction were isolated new phosphoinositides containing manno-oligo-ose as a constituent. These phosphoinositides have been tentatively designated as “Oligomannoinositides.”