Dec. 5 , 1957
METABOLISM OF THIAZOLE-2-Ci4-THIAMINE IN THE [CONTRIBUTION FROM
THE
6321
RAT
DEPARTMENT OF ANIMALSCIENCE, UNIVERSITY OF
ILLINOIS]
Thiamine Metabolism. I. The Metabolism of Thia~ole-Z-C'~-thiaminein Rat's2 BY J. M. IACONO AND B. CONNOR JOHNSON RECEIVED JUNE 17, 1957 This work was undertaken to study the metabolism of radiocarbon-labeled thiamine with particular reference to its metnbolic excretion products. It was found that over 60% of the radioactivity of intraperitoneally injected thiamine was excreted by the rat in the urine and that only small amounts of radioactivity occurred in the feces and in the expired Con. Lt'hilc the majority (approximately 60%) of the radioactivity in the urine was present as unchanged thiamine, it was found that a t least 15 other radioactive metabolites separable by paper chromatography were also present. Three of these have been tentatively identified as thiamine disulfide, thiochrome and the thiazole moiety of thiamine.
Introduction
approximately 500 ml. per minute. lected under toluene.
The urines were col-
The metabolic fate of thiamine has been studied For each rat total radioactivity was estimated in urine, on numerous occasions in various species of animals, feces, expired COz and carcass after a 24-hr. collection pebut little emphasis has been placed on the separa- riod. All measurements of radioactivity were carried out a windowless gas-flow counter (Tracerlab, Inc., Boston, tion and identification of urinary metabolic excre- on Mass., Model No. SC-16) and scaler (Xuclear Chem. and tion products. Thiamine was first determined in Instr. Lab., Chicago, Ill., Model No. 103), and all samples urine by Wang and Harris3 using the thiochrome were corrected for self-absorption by the method of Yankmethod. Kraut and co-workers4 demonstrated wich, et aE." of Chromatograms.-Whatman Nos. 1 and that some of the thiamine in urine is present in the 3 Preparation M M paper were used for paper chromatogrkms. The form of cocarboxylase. The pyrimidine moiety of upper phases of n-butanollacetic acid/water (40: 10: 50) thiamine was reported present in urine by Pollack and sec-butyl alcohol/water, hereafter referred to as nand collaborator^.^ Later Kraut and Wildemann6 butanol and sec-butyl alcohol solvents, respectively, proved suitable for the separation of the known thiamine derivareported thiamine carboxylic acid to be the chief tives and were employed for the separation of the urinary metabolite of thiamine in human urine, with small metabolites of radiothiamine. Descending chromatogamounts of thiochrome also present. Borsook, raphy was used throughout. Identiiication of Thiamine Compounds on Paper Chromaet al. , I injected thiamine labeled with radiosulfur A. Thiochrome Test.-Thiamine, cocarboxylase, into human subjects and found neutral sulfur com- tograms. carboxylic acid and the urinary radiometabolites pounds and inorganic sulfate in urine, and later Mc- thiamine of thiamine were detected by the thiochrome test as modiCarthy and co-workers8 obtained essentially the fied by Kraut, et u Z . ~ Alkaline ferricyanide solution was same results following the administration of S35- sprayed on the paper chromatogram and the chromatograni dried in a current of warm air. Under these conditions thiamine to rats. thiochrome gave a blue fluorescence under the ultraviolet The present paper reports the metabolic fate of lamp. The test is sensitive to .
:
-I:
Pi. 3.-Drawing of radioautograph of metabolite 9 (Table 11). An example of chromatographic purity of a radioactive metabolite after repeated chromatography. Solvent system: water saturated scc-butyl alcohol. Rf value 0.60. Upper line indicates origin. bottom line indicates solvent front.
J. M. IACONO AND B CONNOR JOHNSON
VOl. 79
could be separated from the solids and radioactivity localized in that area; hence, the number of metabolites reported may be less than the number actually excreted. A total of 16 radiometabolites of thiamine are recorded. Of these, 8, shown on Table 11, respond to L . ferment; 36 on a thiamine-deficient medium indicating the presence of the intact thiamine molecule. Four metabolites of the 8 responding to L. ferment; 36 give positive thiochrome tests. One metabolite (spot no. 1, Table 11) which gives a positive thiochrome test behaves like a phosphorylated thiamine derivative, since the corresponding thiochrome compound is not soluble in isobutyl alcohol. On the basis of chromatographic behavior, spot no. 6 corresponds to thiamine and responds positively to thiamine tests. Spot no. 4 takes the same position on the paper chromatogram nl as thiamine disulfide, and spot no. 12 corresponds 0 50 100 to thiochrome and emits the typical bright blue fluorescence of thiochrome under the ultraviolet n-Butanol. lamp. Radioactive spot no. 15 also emits a brilFig. 4.-Schematic chromatogram of rat urine. A liant blue fluorescence similar to that of thiochrome schematic two-dimensional chromatogram of rat urine but does not behave like any known compound. showing the separation of 16 different radioactive metaboSpot no. 16 behaves in the same manner as the lites following the administration of thiazole-2-C14-thiamine. thiazole moiety of thiamine and gives a positive The spot numbers correspond t o those given in Table 11. L. mesenteroides 9135 bioautographic test for thiaThus, spot 6, for example, is thiamine, and spot 12 is thiozole. Figure 4 represents a schematic two-dichrome. mensional radioautograph of the metabolites of clear-cut separations. It became apparent, in the thiamine. The radioactive thiamine used gave, on chroprocess of rechromatographing, that distinct bands turned out on rechromatographing t o be mixtures matography and radioautography, spots correof 2 to 6 radiometabolites. It became necessary, sponding to those for non-labeled thiamine, as given therefore, to rechromatograph each band until no in Table I, and no other spots, thus indicating that further separation occurred. Since two solvent the compound injected was radiochemically pure systems were employed and hence RI values are re- thiamine. Chromatograms of the type illustrated in Fig. 1 corded for each metabolite in the two systems, the final effect was that of a two-dimensional paper were eluted and each band counted. Since subchromatogram (Fig. 4) on enough urine so that sequent work showed the bands obtained to be imthose compounds excreted in small amounts could pure, the data are not presented. However, they be detected a t the same time as purification was did indicate that approximately 60% of the exbeing effected. Unfortunately, in rechromato- creted radioactivity occurred in the thiamine graphing the area a t and just below the origin of band. each sheet, only small amounts of radioactivity URBANA, ILLINOIS
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