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The use of thin-layer chromatography to separate thiamine and related compounds as well as N-methylnicotinamide and related compounds
ANALYTICAL
BIOCHEMISTBY
The
Use
24,
185-191
of Thin-Layer
Thiamine and Related Methylnicotinamide PAUL
P. WARING,
WAYNE
U. S. Army
(1988)
Chromatography Compounds and Related C. GOAD,
AND
Medical Research and Nutrition Fitzsimons General Hospital, Denver, Colorado 80240 Received
October
to Separate as Well as NCompounds ZIGMUND
Z. ZIPORIN
Laboratory,
12, 1967
Studies on the metabolism of thiamine have’largely relied on measuring the amount of the vitamin and its metabolites excreted in the urine (l-9). Some of the identified metabolites of thiamine which are found in the urine are: pyramin (5) ; thiamine disulfide, thiochrome, and thiazole (7) ; and 2-methyl-4-amino-5-pyrimidinecarboxylic acid (PCA) (8, 9). When studies are conducted requiring isolation and identification of metabolites, it is necessary to consider the following compounds, in addition to the metabolites listed above: pyrimidinesulfonic acid, the pyrimidine product of a bisulfite cleavage of thiamine (10) ; a-hydroxyethylthiamine (HET) , a product sometimes found in extracts of bacteria and yeasts (11)) and thus likely to be found following the incubation required in the yeast resynthesis method (12) ; NJ-methylnicotinamide (NMN) , a substance found in urine which remains with thiamine during Decalso column chromatography, and produces a thiochrome-like fluorescent product on oxidation with alkaline ferricyanide (13, 14). It thereby acts as an interfering substance in the pre- and post-oxidation stages of the assay for thiamine in urine. The separation of these substances has been accomplished by the use of paper chromatography (7)) column chromatography followed by paper chromatography (8)) and by thin-layer chromatography (15). Johnson et ‘al. (15) were able to separate thiamine, thiazole, pyrimidinesulfonic acid, ol-hydroxyethylthiamine, and thiochrome by the use of thin-layer chromatography, while GBnshirt and Malzacher (16) and Ishikawa and Katsui (17) reported the separation of thiamine from other vitamins by this method. David and Hirshfeld (18) and Ono and Hara (19) have separated thiamine from its phosphorylated derivatives by thin-layer chromatography. We found the separations reported by Johnson et al. (15) difficult to reproduce because one component of the solvent system, 185 0 1968
by
Arademic
Press
Inc.
186
WARING,
GOAD,
AND
ZIPORIN
pyridine,. absorbs short wavelength ultraviolet light very strongly, thereby masking the components that were not converted to fluorescent products when sprayed with alkaline ferricyanide. In the procedure to be described in this paper, it is possible to separate thiamine, a-hydroxyethylthiamine, N’-methylnicotinamide, thiochrome, pyrimidinesulfonic acid, pyrimidinecarboxyhc acid, and the pyrimidine and thiazoIe moieties of thiamine, as well as the fluorescent oxidation product of N’-methylnicotinamide treated with alkaline ferricyanide. This provides the capability for isolating and identifying most of the substances known to be metabolites of thiamine or associated with thiamine in the various procedures used to study the vitamin. MATERIALS
Thiamine
hydrochloride.
Corp., Cleveland,
AND
METHODS
Purchased
from
Nutritional
Biochemicals
Ohio. Purchased
d-Methyl-5-ethoxymethyl-6-aminopyrimicline.
from Merck
& Co., ‘Rahway, New Jersey. .J-Methyl-5-&hydroxyethyZthiazole.
cu-Hydroqethylthiamine. Prepared procedure of Dr. C. S. Miller (20).
Effect
of pH on R, of Thiamine N’-Methylnicotinamide Eastman Chromagram Aoetonitrile/H~O/formic
Purchased from Merck & Co. in this laboratory according to the
TABLE 1 and Related Compounds and Related Compounds with silica gel (K301R) acid: 40/10/to indicated Solvent
Standard
Thiamine NMN HET Pyrimidine COOH Thiochrome Pyrimidinesulfonic Pyrimidine Thiaeole NMN-&Fe(CN)e NMN-&Fe(CN),
acid
abs. flu.
a A = absorbing. * - = neither absorbing CF = fluorescent.
as
pH
pH
Ultraviolet
2.54
4.03
7.85
0.16 0.31 0.23 0.42 0.31 0.48 0.64 0.85 0.71 0.79
0.04 0.06 0.09 0.21 0.28 0.39 0.65 0.79 0.65 0.75
0.03 0.05 0.06 0.26 0.33 0.46 0.68 0.81 0.67 0.75
nor fluorescing.
as well
2537
Aa A A Fc A A A A F
A
3060
-b F -
A
TLC
OF
THIAMINE
AND
RELATED
187
COMPOUNDS
Pyrirnidinesulfonic acid. Prepared in this laboratory according to the procedure of Goldstein and Brown (10). Pyrimidirmarbo~lic acid. Prepared in this laboratory according to the procedure of Neal and Pearson (9). N’-Methylnicotinamide met&&de (&Fe (CN) ,-treated). About 100 mg of N’-methylnicotinamide methiodide in 5 ml of acidic solution (pH E 2), saturated with NaCl or KU, treated with 3 ml of oxidizing solution (4 ml of 1% K,Fe (CN) 6 to 100 ml with 15% NaOH) , and extracted into 13 ml of isobutyl alcohol. N’-Methylnicotinamide methiodide (&Fe (CN) ,-treated). About 100 eording to the procedure of Huff and Perlzweig (21). Thtichrome. Purchased from Nutritional Biochemicala Corp. Solvents. All solvents used in the chromatography were redistilled. Ethyl alcohol was absolute. Water was distilled, demineralized. Eastman Chromagram .sheets. Type K301R (silica gel with fluorescent indicator). Ultraviolet viewing box. BLE Spectroline, model C-3F, Spectronics Corp., Westbury, L. I., New York, equipped with short wavelength (2537 A) and long wavelength (3660B) lamps.
SOLYENT
FRONT
u 0
FIG. 1. One-dimensional system : Acetonitrile/H~O/formic methylnicotinamide. HET
0
0 0
8
o
chromatogram on Eastman Chromagram acid : 4O/lO/pH adjusted to = a-hydroxyethyBhiamine.
K3OlR. 2.54. NMN
Sohent = N’-
188
WARING,
GOAD,
AND
ZIPORIN
FIQ. 2 Two-dimensional chromatogram on Eastman Chromagram K301R. Phase 1: Acetonitrile/HIO/formic acid : 40/10/pH adjusted to 4.03. Phase 2 : Acetonitrile/ HzO/formic acid: 40/10/pH adjusted to 2.58.
Methods
Standards were prepared in aqueous solutions at concentrations of 0.54 mg/ml, of which 0.2-2 X was spotted on the Chromagram sheets for one- or two-dimensional ascending chromatography. Solvent mixtures were allowed to equilibrate for 15 min in covered chromatography ja.rs before placing the Chromagram sheets in the tanks for development. The solvent front required 80 min to ascend 16-18 cm. The plates were then removed and air-dried rapidly with a hair dryer. Detection of the spots was accomplished in the ultraviolet viewing box using the short wavelength (2537A) for revealing the absorbing compounds, and the 3660B wavelength for detecting the fluorescent compounds. The latter compounds were derived from thiamine, a-hydroxyethylthiamine, and N’-methylnicotinamide when they were sprayed with alkaline ferricyanide. RESULTS
AND
DISCUSSION
From the Rf values presented in Table 1, it is apparent that the more acidic the solvent system, the greater is the movement of substances with
TLC
OF
THIAMINE
AND
RELATED
189
COMPOUNDS
Rf values of 0.16-0.42, while those substances with Rf values >0.48 are not markedly affected by changes in pH of the solvent systems. The final procedure which was adopted permitted the resolution of all substances tested except for N’-methylnicotinamide and thiochrome (Fig..l) These could be distinguished on the Chromagram without further treatment since with short wavelength ultraviolet light N’-methylnicotinamide absorbed but did not fluoresce, while thiochrome neither absorbed nor fluoresced. With long wavelength ultraviolet light, IV’-methylincotinamide neither absorbed nor fluoresced, while thiochrome fluoresced intensely with a light blue color. For quantitative determination of each, it was possible to effect a separation with two-dimensional chromatography (Fig. 2)) while at the same time obtaining good resolution of other compounds related to thiamine. An unidentified absorbing spot was formed along the fluorescent product during the treatment of N’-methylnicotinamide with alkaline potassium ferricyanide (Table 2, Fig. 1). When it became apparent that acidic solvent systems provided separations of the compounds with the smaller Rjs, various acidic components TABLE 2 RI Values of Standards in Various Solvent Systems Using One-Dimensional Chromatography Solvent Standard
Thiamine NMN HET Pyrimidine COOH Thiochrome Pyrimidinesulfonic acid Pyrimidine Thiazole NMN-KJFe(CN)e abs. NMN-&Fe(CN)s flu. Approx. time required for solvent to travel 16-18 cm, min
system
A
B
C
Da
En
F&
Go
0.16 0.31 0.23 0.42 0.31 0.48 0.64 0.85 0.71 0.79 80
0.26 0.32
0.05 0.04
0.15 0.23
0.26 0.14
0.04 0.04
0.46 0.26
0.19 0.35
0.31
0.71 0.87
0.71 0.85
0.71 0.78
0.26 0.56 0.28 0.71 0.80
0.03 0.15 0.07 0.57 0.84
0.10 0.00 0.47 0.60 0.63 0.68 0.83 0.91
75
80
80
90
60
180
Solvent systems: A.. Acetonitrile/&O/formic acid: 40/1O/pH adjusted to 2.54. B.. Acetonitrile/isobutyric acid/H20: 40/20/5. ..s.. ‘. C. Acetonitrile/tertamy1 alcohol/HaO: 46/10/5. D. Acetonitrile/benzoic acid/H&: 80/500 mg/20. E. Acetonitrile/phenol/H~O: 40/10/10. F. Acetonit+le/phenol: 40/10. G. Pyridine/ieobutanol/HzO: 40/10/10 (Johnson el al., 15). a Not easily visualized under ultraviolet.
190
WARING,
GOAD,
AND
ZIPORIN
were tried. The results obtained with these systems are presented in Table 2. With acetonitrile as one component of the system and water in all but one, acidic pH’s were sought using benzoic acid, phenol, isobutyric acid and formic acid. The pyridine/isobutanol/water (4/1/l) was t.he same as that employed by Johnson et al. (15). We found the solvent systems containing benzoic acid, phenol, and pyridine unsatisfactory because of the strong ultraviolet absorption at 2537 A, thus masking the absorbing spots on the chromatogram. The procedure outlined in this paper provides good resolution of compounds related to thiamine and N’-methylnicotinamide, while at the same time allowing for visualization as absorbing or fluorescing spots. The pH of the solvent systems are in the range that would ensure stability of t,he tihromatographed compounds. It should be possible to use this procedure as a tool to follow thiamine metabolism in a test organism. SUMMARY
A thin-layer chromatographic procedure is presented which can separate thiamine, a-hydroxyethylthiamine, thiochrome, pyrimidinesulfonic acid, pyrimidinecarboxylic acid, the pyrimidine and thiazole moieties of thiamine, N’-methylnicotinamide, and the fluorescing and absorbing product derived from the oxidation of N’-methylnicotinamide by alkaline ferricyanide. The ability to separate these substances should provide a useful tool for studying the methabolism of thiamine. REFERENCES 1. ELBOM, K. O’S., REINHOLD, J. G., NICHOLSON, J. T. L., AND CHORNOCK, C., Am. J. Med. Sci. 203, 569 (1942). 2. HATHAWAY, M. L., AND STROM, J. E., J. Nutrition 32, 1 (1946). 3. OLDHAM, H. G., DAVIS, M. V., AND ROBERTS, L. J., J. Nutrition 32, 163 (1946). 4. DAUM, K., TUTTLE, W. W., WILSON, M., AND RHODES, H., J. Am. Dietet. Assoc. 24, 1049 (1948). 5. MICKELSEN, O., CASTER, W. O., AND KEYS, A., J. Biol. Chem. 168, 415 (1947). 6. ZIPORIN, Z. Z., NUNES, W. T., POWELL, R. C., WARINQ, P. P., AND SAUBEXLLICH, H. E., J. Nutrition 85,287 (1965). 7. IACONO, J. M., AND JOHNSON, B. C., J. Am. Chem. Sot. 79, 6321 (1957). 8. NEAL, R. A., AND PEARSON, W. N., J. Nutrition 83,343 (1$X). 9. NEAL, R. A., AND PEARSON, W. N., J. *Nutdon 83,351. (1964). G. A? AND -BROWN., G. M., Arch. Biochem. Biophys. 103, 449 (1963). 10. GOLDSTE~, 11. CARLSON, G. fi., AN&BROWN, Gi‘M., J. Biol. Chem. 236,2099 (1961). 12. ZIPORIN, Z. Z., .BI&E; E., HOLLAND, D. C., AND BIERMAN, E. L., Anat. Biochem. 3, 1 (1962). 13. NAJJAR, V. A., AND WOOD, R. W., Proc. Sot. Exptl. Biol. Med. 44, 386 (1940). 14. HUFF, J. W., AND PERLZWEIQ, W. A., Science 97, 538 (1943). 15. JOHNSON, D. B., HOWELLS, D. J., AND GOODWIN, T. W., Biochem. J. 98, 30 (1966). 16. G&SHIRT, H., AND MALZACHER, A., Nuturwissenschujten 47, 279 (1960). S., AND KATSUI, G., Bitamin 29,203 (1964). 17. I~HIEAWA,
TLC
OF
THIAMINE
AND
RELATED
COMPOUNDS
191
18. DAVID, S., AND HIRSHFELD, H., Bull. Sot. Chim. France 1011. (1963). 19. ONO, T., AND HABA, M., Bitamin 33, 512 (1966). 20. Personal communication from Dr. James M. Sprague, Merck, Sharp & Dohme, West Point, Pa., describing the method of synthesis by Dr. Charles S. Miller. 21. HUFF, J. W., AND PERLZWEIC, W. A., J. Biol. Chem. 150, 395 (1943).
BIOCHEMISTBY
The
Use
24,
185-191
of Thin-Layer
Thiamine and Related Methylnicotinamide PAUL
P. WARING,
WAYNE
U. S. Army
(1988)
Chromatography Compounds and Related C. GOAD,
AND
Medical Research and Nutrition Fitzsimons General Hospital, Denver, Colorado 80240 Received
October
to Separate as Well as NCompounds ZIGMUND
Z. ZIPORIN
Laboratory,
12, 1967
Studies on the metabolism of thiamine have’largely relied on measuring the amount of the vitamin and its metabolites excreted in the urine (l-9). Some of the identified metabolites of thiamine which are found in the urine are: pyramin (5) ; thiamine disulfide, thiochrome, and thiazole (7) ; and 2-methyl-4-amino-5-pyrimidinecarboxylic acid (PCA) (8, 9). When studies are conducted requiring isolation and identification of metabolites, it is necessary to consider the following compounds, in addition to the metabolites listed above: pyrimidinesulfonic acid, the pyrimidine product of a bisulfite cleavage of thiamine (10) ; a-hydroxyethylthiamine (HET) , a product sometimes found in extracts of bacteria and yeasts (11)) and thus likely to be found following the incubation required in the yeast resynthesis method (12) ; NJ-methylnicotinamide (NMN) , a substance found in urine which remains with thiamine during Decalso column chromatography, and produces a thiochrome-like fluorescent product on oxidation with alkaline ferricyanide (13, 14). It thereby acts as an interfering substance in the pre- and post-oxidation stages of the assay for thiamine in urine. The separation of these substances has been accomplished by the use of paper chromatography (7)) column chromatography followed by paper chromatography (8)) and by thin-layer chromatography (15). Johnson et ‘al. (15) were able to separate thiamine, thiazole, pyrimidinesulfonic acid, ol-hydroxyethylthiamine, and thiochrome by the use of thin-layer chromatography, while GBnshirt and Malzacher (16) and Ishikawa and Katsui (17) reported the separation of thiamine from other vitamins by this method. David and Hirshfeld (18) and Ono and Hara (19) have separated thiamine from its phosphorylated derivatives by thin-layer chromatography. We found the separations reported by Johnson et al. (15) difficult to reproduce because one component of the solvent system, 185 0 1968
by
Arademic
Press
Inc.
186
WARING,
GOAD,
AND
ZIPORIN
pyridine,. absorbs short wavelength ultraviolet light very strongly, thereby masking the components that were not converted to fluorescent products when sprayed with alkaline ferricyanide. In the procedure to be described in this paper, it is possible to separate thiamine, a-hydroxyethylthiamine, N’-methylnicotinamide, thiochrome, pyrimidinesulfonic acid, pyrimidinecarboxyhc acid, and the pyrimidine and thiazoIe moieties of thiamine, as well as the fluorescent oxidation product of N’-methylnicotinamide treated with alkaline ferricyanide. This provides the capability for isolating and identifying most of the substances known to be metabolites of thiamine or associated with thiamine in the various procedures used to study the vitamin. MATERIALS
Thiamine
hydrochloride.
Corp., Cleveland,
AND
METHODS
Purchased
from
Nutritional
Biochemicals
Ohio. Purchased
d-Methyl-5-ethoxymethyl-6-aminopyrimicline.
from Merck
& Co., ‘Rahway, New Jersey. .J-Methyl-5-&hydroxyethyZthiazole.
cu-Hydroqethylthiamine. Prepared procedure of Dr. C. S. Miller (20).
Effect
of pH on R, of Thiamine N’-Methylnicotinamide Eastman Chromagram Aoetonitrile/H~O/formic
Purchased from Merck & Co. in this laboratory according to the
TABLE 1 and Related Compounds and Related Compounds with silica gel (K301R) acid: 40/10/to indicated Solvent
Standard
Thiamine NMN HET Pyrimidine COOH Thiochrome Pyrimidinesulfonic Pyrimidine Thiaeole NMN-&Fe(CN)e NMN-&Fe(CN),
acid
abs. flu.
a A = absorbing. * - = neither absorbing CF = fluorescent.
as
pH
pH
Ultraviolet
2.54
4.03
7.85
0.16 0.31 0.23 0.42 0.31 0.48 0.64 0.85 0.71 0.79
0.04 0.06 0.09 0.21 0.28 0.39 0.65 0.79 0.65 0.75
0.03 0.05 0.06 0.26 0.33 0.46 0.68 0.81 0.67 0.75
nor fluorescing.
as well
2537
Aa A A Fc A A A A F
A
3060
-b F -
A
TLC
OF
THIAMINE
AND
RELATED
187
COMPOUNDS
Pyrirnidinesulfonic acid. Prepared in this laboratory according to the procedure of Goldstein and Brown (10). Pyrimidirmarbo~lic acid. Prepared in this laboratory according to the procedure of Neal and Pearson (9). N’-Methylnicotinamide met&&de (&Fe (CN) ,-treated). About 100 mg of N’-methylnicotinamide methiodide in 5 ml of acidic solution (pH E 2), saturated with NaCl or KU, treated with 3 ml of oxidizing solution (4 ml of 1% K,Fe (CN) 6 to 100 ml with 15% NaOH) , and extracted into 13 ml of isobutyl alcohol. N’-Methylnicotinamide methiodide (&Fe (CN) ,-treated). About 100 eording to the procedure of Huff and Perlzweig (21). Thtichrome. Purchased from Nutritional Biochemicala Corp. Solvents. All solvents used in the chromatography were redistilled. Ethyl alcohol was absolute. Water was distilled, demineralized. Eastman Chromagram .sheets. Type K301R (silica gel with fluorescent indicator). Ultraviolet viewing box. BLE Spectroline, model C-3F, Spectronics Corp., Westbury, L. I., New York, equipped with short wavelength (2537 A) and long wavelength (3660B) lamps.
SOLYENT
FRONT
u 0
FIG. 1. One-dimensional system : Acetonitrile/H~O/formic methylnicotinamide. HET
0
0 0
8
o
chromatogram on Eastman Chromagram acid : 4O/lO/pH adjusted to = a-hydroxyethyBhiamine.
K3OlR. 2.54. NMN
Sohent = N’-
188
WARING,
GOAD,
AND
ZIPORIN
FIQ. 2 Two-dimensional chromatogram on Eastman Chromagram K301R. Phase 1: Acetonitrile/HIO/formic acid : 40/10/pH adjusted to 4.03. Phase 2 : Acetonitrile/ HzO/formic acid: 40/10/pH adjusted to 2.58.
Methods
Standards were prepared in aqueous solutions at concentrations of 0.54 mg/ml, of which 0.2-2 X was spotted on the Chromagram sheets for one- or two-dimensional ascending chromatography. Solvent mixtures were allowed to equilibrate for 15 min in covered chromatography ja.rs before placing the Chromagram sheets in the tanks for development. The solvent front required 80 min to ascend 16-18 cm. The plates were then removed and air-dried rapidly with a hair dryer. Detection of the spots was accomplished in the ultraviolet viewing box using the short wavelength (2537A) for revealing the absorbing compounds, and the 3660B wavelength for detecting the fluorescent compounds. The latter compounds were derived from thiamine, a-hydroxyethylthiamine, and N’-methylnicotinamide when they were sprayed with alkaline ferricyanide. RESULTS
AND
DISCUSSION
From the Rf values presented in Table 1, it is apparent that the more acidic the solvent system, the greater is the movement of substances with
TLC
OF
THIAMINE
AND
RELATED
189
COMPOUNDS
Rf values of 0.16-0.42, while those substances with Rf values >0.48 are not markedly affected by changes in pH of the solvent systems. The final procedure which was adopted permitted the resolution of all substances tested except for N’-methylnicotinamide and thiochrome (Fig..l) These could be distinguished on the Chromagram without further treatment since with short wavelength ultraviolet light N’-methylnicotinamide absorbed but did not fluoresce, while thiochrome neither absorbed nor fluoresced. With long wavelength ultraviolet light, IV’-methylincotinamide neither absorbed nor fluoresced, while thiochrome fluoresced intensely with a light blue color. For quantitative determination of each, it was possible to effect a separation with two-dimensional chromatography (Fig. 2)) while at the same time obtaining good resolution of other compounds related to thiamine. An unidentified absorbing spot was formed along the fluorescent product during the treatment of N’-methylnicotinamide with alkaline potassium ferricyanide (Table 2, Fig. 1). When it became apparent that acidic solvent systems provided separations of the compounds with the smaller Rjs, various acidic components TABLE 2 RI Values of Standards in Various Solvent Systems Using One-Dimensional Chromatography Solvent Standard
Thiamine NMN HET Pyrimidine COOH Thiochrome Pyrimidinesulfonic acid Pyrimidine Thiazole NMN-KJFe(CN)e abs. NMN-&Fe(CN)s flu. Approx. time required for solvent to travel 16-18 cm, min
system
A
B
C
Da
En
F&
Go
0.16 0.31 0.23 0.42 0.31 0.48 0.64 0.85 0.71 0.79 80
0.26 0.32
0.05 0.04
0.15 0.23
0.26 0.14
0.04 0.04
0.46 0.26
0.19 0.35
0.31
0.71 0.87
0.71 0.85
0.71 0.78
0.26 0.56 0.28 0.71 0.80
0.03 0.15 0.07 0.57 0.84
0.10 0.00 0.47 0.60 0.63 0.68 0.83 0.91
75
80
80
90
60
180
Solvent systems: A.. Acetonitrile/&O/formic acid: 40/1O/pH adjusted to 2.54. B.. Acetonitrile/isobutyric acid/H20: 40/20/5. ..s.. ‘. C. Acetonitrile/tertamy1 alcohol/HaO: 46/10/5. D. Acetonitrile/benzoic acid/H&: 80/500 mg/20. E. Acetonitrile/phenol/H~O: 40/10/10. F. Acetonit+le/phenol: 40/10. G. Pyridine/ieobutanol/HzO: 40/10/10 (Johnson el al., 15). a Not easily visualized under ultraviolet.
190
WARING,
GOAD,
AND
ZIPORIN
were tried. The results obtained with these systems are presented in Table 2. With acetonitrile as one component of the system and water in all but one, acidic pH’s were sought using benzoic acid, phenol, isobutyric acid and formic acid. The pyridine/isobutanol/water (4/1/l) was t.he same as that employed by Johnson et al. (15). We found the solvent systems containing benzoic acid, phenol, and pyridine unsatisfactory because of the strong ultraviolet absorption at 2537 A, thus masking the absorbing spots on the chromatogram. The procedure outlined in this paper provides good resolution of compounds related to thiamine and N’-methylnicotinamide, while at the same time allowing for visualization as absorbing or fluorescing spots. The pH of the solvent systems are in the range that would ensure stability of t,he tihromatographed compounds. It should be possible to use this procedure as a tool to follow thiamine metabolism in a test organism. SUMMARY
A thin-layer chromatographic procedure is presented which can separate thiamine, a-hydroxyethylthiamine, thiochrome, pyrimidinesulfonic acid, pyrimidinecarboxylic acid, the pyrimidine and thiazole moieties of thiamine, N’-methylnicotinamide, and the fluorescing and absorbing product derived from the oxidation of N’-methylnicotinamide by alkaline ferricyanide. The ability to separate these substances should provide a useful tool for studying the methabolism of thiamine. REFERENCES 1. ELBOM, K. O’S., REINHOLD, J. G., NICHOLSON, J. T. L., AND CHORNOCK, C., Am. J. Med. Sci. 203, 569 (1942). 2. HATHAWAY, M. L., AND STROM, J. E., J. Nutrition 32, 1 (1946). 3. OLDHAM, H. G., DAVIS, M. V., AND ROBERTS, L. J., J. Nutrition 32, 163 (1946). 4. DAUM, K., TUTTLE, W. W., WILSON, M., AND RHODES, H., J. Am. Dietet. Assoc. 24, 1049 (1948). 5. MICKELSEN, O., CASTER, W. O., AND KEYS, A., J. Biol. Chem. 168, 415 (1947). 6. ZIPORIN, Z. Z., NUNES, W. T., POWELL, R. C., WARINQ, P. P., AND SAUBEXLLICH, H. E., J. Nutrition 85,287 (1965). 7. IACONO, J. M., AND JOHNSON, B. C., J. Am. Chem. Sot. 79, 6321 (1957). 8. NEAL, R. A., AND PEARSON, W. N., J. Nutrition 83,343 (1$X). 9. NEAL, R. A., AND PEARSON, W. N., J. *Nutdon 83,351. (1964). G. A? AND -BROWN., G. M., Arch. Biochem. Biophys. 103, 449 (1963). 10. GOLDSTE~, 11. CARLSON, G. fi., AN&BROWN, Gi‘M., J. Biol. Chem. 236,2099 (1961). 12. ZIPORIN, Z. Z., .BI&E; E., HOLLAND, D. C., AND BIERMAN, E. L., Anat. Biochem. 3, 1 (1962). 13. NAJJAR, V. A., AND WOOD, R. W., Proc. Sot. Exptl. Biol. Med. 44, 386 (1940). 14. HUFF, J. W., AND PERLZWEIQ, W. A., Science 97, 538 (1943). 15. JOHNSON, D. B., HOWELLS, D. J., AND GOODWIN, T. W., Biochem. J. 98, 30 (1966). 16. G&SHIRT, H., AND MALZACHER, A., Nuturwissenschujten 47, 279 (1960). S., AND KATSUI, G., Bitamin 29,203 (1964). 17. I~HIEAWA,
TLC
OF
THIAMINE
AND
RELATED
COMPOUNDS
191
18. DAVID, S., AND HIRSHFELD, H., Bull. Sot. Chim. France 1011. (1963). 19. ONO, T., AND HABA, M., Bitamin 33, 512 (1966). 20. Personal communication from Dr. James M. Sprague, Merck, Sharp & Dohme, West Point, Pa., describing the method of synthesis by Dr. Charles S. Miller. 21. HUFF, J. W., AND PERLZWEIC, W. A., J. Biol. Chem. 150, 395 (1943).