Glucosamine Acetoacetate Condensate II. Isolation and Identification from Human Diabetic Urines

Abstract

Previous work in our laboratories indicated that the condensation product of D-glucosamine and ethyl acetoacetate, ethyl 2-methyl-5 -( D-arabinotetrahydroxybutyl) -3 -py r r ole carboxylate (pyrrole condensate) ( 1 ) , acts as an antialloxan-diabetes factor (2). Also chromatographic evidence for the presence of the pyrrole condensate in the human diabetic urine was found. Its isolation and identification became the next goal in our research studies.Materials and Methods At least 75 ml of urine is refrigerated for 24 hr to cause precipitation of inorganic salts and after filtration, it is concentrated by blowing cold air through a semipermeable membrane filled with the specimen. The yield of dry material is approximately 1-2 g. The dry material is dissolved in 50% alcohol and applied to thinlayer chromatography (Eastman silica gel), both one dimensional (butanol-acetic acidwater, 4 : 1 : 5) and two dimensional (chloroform-acetic acid-water, 2 : 1 : 1 and 20% KC1). The isolated pyrrole condensate was eluted with 95% alcohol and used in the identification experiments after drying. The yield of isolated pyrrole condensate is approximately 1.5-2.0 mg. This material was used for the instrumentd analyses.The KBr pellets (1 mg of sample to 10 mg of KBr) were prepared from the dried isolated pyrrole condensate for infrared studies. Solutions of the isolated pyrrole condensate in absolute ethanol were used for the ultraviolet absorption studies.For the gas chromatography analysis, the dried isolated pyrrole condensate and the known pyrrde condensate were treated with 1 Supported by a grant from the Research Corporation.Tri-Sil (trimethylcholorosilane from Pierce Chemical Company) in order to make the more volatile trimethysilyl ether derivations to be separated on a 5% SE 52 on 60/70 Anakrom ABS column under the following conditions: detector temperature, 2 50" ; injector temperature, 150"; flow rate, 60 ml/min helium; current, 125 mA; polarity, positive; attenuator, 2 ; and sample size, 40 pl. Column temperature was programmed to increase from 125" at the rate of 4.5'3Jmin.Results. The presence of pyrrole condensate in 13 out of 14 diabetic urines was established using ultraviolet, infrared spectrophotometry, and gas chromatography. Of the 14 urines from patients with diabetes mellitusg isolated pyrrole condensate from 6 urines show characteristic absorption maxima of 230 and 265 mp which agrees with the data obtained for the known condensate, and six additional urines show characteristic absorption maxima of 225 and 275 mp which agrees with that obtained for the salt form of the condensate. On the other hand, the condensate was not found in any of 14 normal urines. (Table I, Cdumn 1 ) . A comparison of a typical curve obtained from the UV absorption characteristic of the known pyrrole condensate as compared with the isolated one from a diabetic urine is shown in Fig. 1A and B ; and the curves for the known and isolated salts are compared in Fig. 1C and D. Both the shape of the curve and the absorption maxima correspond n...

Glucosamine Acetoacetate Condensate Iii. Its Effect Upon Serum Insulin and Glucose Uptake by Muscle and Liver

“…Presently, the only indirect evidences that we have stabilized connecting pyrrolecondensate with human diabetes are: Its isolation from human diabetic urine (Padron et al, 1969) and the presence of larger amounts of bound glucosamine in the diabetic urine than in the normal urine (Padron et al, 1967). Also glucosamine, one of the components of the pyrrole-condensate, has been found by Stary et al (1950) to be subject to changes in both diabetes mellitus and experimental alloxan diabetes.…”

“…Previous work in our laboratories indicated that the condensation product of D-:glucosamine and ethylacetoacetate, ethyl-2-methyl-5-(D-arabino tetrahydroxybutyl)-3-pyrrole carboxylate (pyrrole-condensate) (Gonzalez and Sanchez, 1965) acts as an antialloxan-diabetes factor in rats (Padron, Sulman and Lewis, 1967) and can be isolated and identified from human diabetic urine (Padron, Chittenden and Lewis, 1969).…”