- Email: [email protected]
[10] Fluorometric determination of thiamine and its mono-, di-, and triphosphate esters
58
'
THIAMINE:
PHOSPHATES
AND
ANALOGS
[10]
[10] F l u o r o m e t r i c D e t e r m i n a t i o n o f T h i a m i n e a n d I t s M o n o - , Di-, a n d T r i p h o s p h a t e E s t e r s
By H. K. PENTTINEN Thiamine, when oxidized in an alkaline medium, is converted to the fluorescent thiochrome derivative. This reaction is used in the quantitative estimation of thiamine and its phosphate esters. However, equimolar amounts of these compounds produce unequal intensities of thiochrome fluorescence. Furthermore, addition of ethanol to the oxidation medium favors the production of thiochrome. These observations 1 have made it necessary to modify the previous method of Lewin and Wei. 2
Reagents Ethanol, 50% (v/v) Alkaline ferricyanide [hexacyanoferrate(III)] (15 ml of 15% NaOH and 1 ml of 2% potassium ferricyanide) Hydrogen peroxide solution, 30% Quinine sulfate (0.01% quinine sulfate in 0. I M H2SO4) Thiamine standard solution, 0.3 mM, U.S.P. Reference Standard Benzenesulfonyl chloride reagent, diluted with ethanol (1:6, v/v), (Koch-Light Laboratories Ltd., England) Assay Procedure A 5-/~I sample of thiamine or its phosphate ester is added to 3 ml of 50% ethanol and shaken. After a few minutes, 0.5 ml of alkaline fcrricyanide is added, and the mixture is agitated for 2 min. Then I0/~l of 30~ H~Os is added to destroy the yellow color of fcrricyanide, and the fluorescence can be measured. Fluorometric measurements are conducted in a Farrand fluorometer A 4 with PC Corning Filtersnumbers 7-37 as primary filterand 3-73 and 5-60 as secondary filters,respectively, or in a Zeiss P M Q II spectrophotometer fittedwith a Z F M 4 fluorometer attachment. The excitation wavelength is 365 nm, and the emission m a x i m u m is 430 am.
The background fluorescence is determined by omitting the thiamine compound. It is also estimated by dissolving the sample in 2.8 ml of 50% ethanol, then adding 0.2 ml of benzenesulfonyl chloride reagent and stirring the mixture. Determination is then continued as described before. i H , K . P e n t t i n e n , Acta Chem. Scand. B 3 0 , 6 5 9 (1976). 2 L . M . L e w i n a n d R . W e i , Anal. Biochem. 16, 2 9 (1966).
METHODS IN ENZYMOLOGY, VOL 62
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved, ISBN 0-12-181962-0
[11]
S E P A R A T I O N OF T H I A M I N E
ESTERS ON S E P H A D E X
59
Correction of the Deficient Thiochrome Formation Because in this oxidation medium, 74% of thiamine and thiamine monophosphate, 85% of thiamine diphosphate, and 93% of thiamine triphosphate are converted to thiochrome, the fluorescence values have to be corrected. When thiamine is used as standard, the fluorescence reading for thiamine monophosphate needs no correction, but the readings for thiamine di- and triphosphate have to be multiplied by 0.87 and 0.80, respectively) These correction factors have been shown to be constant and independent of the concentration of thiamine compounds.
[ 11] S e p a r a t i o n o f T h i a m i n e P h o s p h o r i c on Sephadex Cation Exchanger
Esters
B y JULIA M. PARKHOMENKO, ALLA A. RYBINA,
and
ASKAR G.
KHALMURADOV
The main problem in estimating thiamine phosphoric esters in biological material is their quantitative separation.~-4 Separating these pure compounds from their mixture presents no difficulties. Several methods have been suggested, and the more suitable appear to be electrophoresis on cellulose polyacetate strips 5 and chromatography on various ion-exchange resins.°'7 However, high concentrations of inorganic salts and other impurities complicate greatly the separation of thiamine compounds by the above-mentioned methods. Rindi and de Giuseppe s and Koike et al. 9 succeeded in purifying a mixture of thiamine esters on a column packed with active charcoal treated with cholesteryl stearate ° prior to their separation on ion-exchange resins, such as Dowex l-X8 s and Dowex l-X4. 9 Sephadex appeared to be a suitable material for desalting (Sephadex G-25) TM and partial separation (Sephadex G-10) H of the thiamines. However, 1 A. Rossi-Fanelli, Science 116, 711 (1952). 2 W. Bartley, Biochem. J. 56, 379 (1954). 3 A. A. Rybina, in " V i t a m i n s , " Vol. 4, p. 10. Akad. N a n k U S S R , Kiev, 1959. 4 y . V. K h m e l e v s k i i , Vopr. Med. Khim. 8, 542 (1962). 5 y . Itokawa and J. R. Cooper, this series, Vol. 18A, p. 91. 6 D. Siliprandi and N. Siliprandi, Biochim. Biophys. Acta 14, 52 (1954). 7 L. de Giuseppe and G. Rindi, J. Chromatogr. 1,545 (1958). s G. Rindi and L. de Giuseppe, Biochem. J. 78, 602 (1961). 9 H. Koike, T. W a d a , a n d H. Minakami, J. Biochem. (Tokyo) 62, 492 (1967). 10 L. W i l d e m a n n , Z. Klin. Chem. Klin. Biochem. 7 , 5 0 9 (1969); cited in Nutr. Abstr. Rev. 40, 832 (1970). ~1 T. N i s h i m u n e , M. Abe, and R. H a y a s h i , Biochim. Biophys. Acta 279, 527 (1972).
METHODS IN ENZYMOLOGY, VOL. 62
Copyright © 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-0
'
THIAMINE:
PHOSPHATES
AND
ANALOGS
[10]
[10] F l u o r o m e t r i c D e t e r m i n a t i o n o f T h i a m i n e a n d I t s M o n o - , Di-, a n d T r i p h o s p h a t e E s t e r s
By H. K. PENTTINEN Thiamine, when oxidized in an alkaline medium, is converted to the fluorescent thiochrome derivative. This reaction is used in the quantitative estimation of thiamine and its phosphate esters. However, equimolar amounts of these compounds produce unequal intensities of thiochrome fluorescence. Furthermore, addition of ethanol to the oxidation medium favors the production of thiochrome. These observations 1 have made it necessary to modify the previous method of Lewin and Wei. 2
Reagents Ethanol, 50% (v/v) Alkaline ferricyanide [hexacyanoferrate(III)] (15 ml of 15% NaOH and 1 ml of 2% potassium ferricyanide) Hydrogen peroxide solution, 30% Quinine sulfate (0.01% quinine sulfate in 0. I M H2SO4) Thiamine standard solution, 0.3 mM, U.S.P. Reference Standard Benzenesulfonyl chloride reagent, diluted with ethanol (1:6, v/v), (Koch-Light Laboratories Ltd., England) Assay Procedure A 5-/~I sample of thiamine or its phosphate ester is added to 3 ml of 50% ethanol and shaken. After a few minutes, 0.5 ml of alkaline fcrricyanide is added, and the mixture is agitated for 2 min. Then I0/~l of 30~ H~Os is added to destroy the yellow color of fcrricyanide, and the fluorescence can be measured. Fluorometric measurements are conducted in a Farrand fluorometer A 4 with PC Corning Filtersnumbers 7-37 as primary filterand 3-73 and 5-60 as secondary filters,respectively, or in a Zeiss P M Q II spectrophotometer fittedwith a Z F M 4 fluorometer attachment. The excitation wavelength is 365 nm, and the emission m a x i m u m is 430 am.
The background fluorescence is determined by omitting the thiamine compound. It is also estimated by dissolving the sample in 2.8 ml of 50% ethanol, then adding 0.2 ml of benzenesulfonyl chloride reagent and stirring the mixture. Determination is then continued as described before. i H , K . P e n t t i n e n , Acta Chem. Scand. B 3 0 , 6 5 9 (1976). 2 L . M . L e w i n a n d R . W e i , Anal. Biochem. 16, 2 9 (1966).
METHODS IN ENZYMOLOGY, VOL 62
Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved, ISBN 0-12-181962-0
[11]
S E P A R A T I O N OF T H I A M I N E
ESTERS ON S E P H A D E X
59
Correction of the Deficient Thiochrome Formation Because in this oxidation medium, 74% of thiamine and thiamine monophosphate, 85% of thiamine diphosphate, and 93% of thiamine triphosphate are converted to thiochrome, the fluorescence values have to be corrected. When thiamine is used as standard, the fluorescence reading for thiamine monophosphate needs no correction, but the readings for thiamine di- and triphosphate have to be multiplied by 0.87 and 0.80, respectively) These correction factors have been shown to be constant and independent of the concentration of thiamine compounds.
[ 11] S e p a r a t i o n o f T h i a m i n e P h o s p h o r i c on Sephadex Cation Exchanger
Esters
B y JULIA M. PARKHOMENKO, ALLA A. RYBINA,
and
ASKAR G.
KHALMURADOV
The main problem in estimating thiamine phosphoric esters in biological material is their quantitative separation.~-4 Separating these pure compounds from their mixture presents no difficulties. Several methods have been suggested, and the more suitable appear to be electrophoresis on cellulose polyacetate strips 5 and chromatography on various ion-exchange resins.°'7 However, high concentrations of inorganic salts and other impurities complicate greatly the separation of thiamine compounds by the above-mentioned methods. Rindi and de Giuseppe s and Koike et al. 9 succeeded in purifying a mixture of thiamine esters on a column packed with active charcoal treated with cholesteryl stearate ° prior to their separation on ion-exchange resins, such as Dowex l-X8 s and Dowex l-X4. 9 Sephadex appeared to be a suitable material for desalting (Sephadex G-25) TM and partial separation (Sephadex G-10) H of the thiamines. However, 1 A. Rossi-Fanelli, Science 116, 711 (1952). 2 W. Bartley, Biochem. J. 56, 379 (1954). 3 A. A. Rybina, in " V i t a m i n s , " Vol. 4, p. 10. Akad. N a n k U S S R , Kiev, 1959. 4 y . V. K h m e l e v s k i i , Vopr. Med. Khim. 8, 542 (1962). 5 y . Itokawa and J. R. Cooper, this series, Vol. 18A, p. 91. 6 D. Siliprandi and N. Siliprandi, Biochim. Biophys. Acta 14, 52 (1954). 7 L. de Giuseppe and G. Rindi, J. Chromatogr. 1,545 (1958). s G. Rindi and L. de Giuseppe, Biochem. J. 78, 602 (1961). 9 H. Koike, T. W a d a , a n d H. Minakami, J. Biochem. (Tokyo) 62, 492 (1967). 10 L. W i l d e m a n n , Z. Klin. Chem. Klin. Biochem. 7 , 5 0 9 (1969); cited in Nutr. Abstr. Rev. 40, 832 (1970). ~1 T. N i s h i m u n e , M. Abe, and R. H a y a s h i , Biochim. Biophys. Acta 279, 527 (1972).
METHODS IN ENZYMOLOGY, VOL. 62
Copyright © 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181962-0