Thiamin compounds

Thiamin compounds can also be measured by HPLC, A clever techniejue has been used to facilitate the detection of thiamin compounds immediately after they have completed their chromatographic separation by HPLC, The mixture of un-separated compounds is exposed to conditions expected to provoke the conversion of thiamin to its thiochrome derivative, and then applied to the HPLC column (Tallakesen ef rt/., 1997 Rindi and Laforenza, 1997), This approach,called "pre-column derivatization/ is used widely by chemists and biochemists during the separation and detection of many compounds. 

Kawasaki, C. Modified thiamine compounds. Vitamins and Hormones 21,69 (1963). 

Thiamin (vitamin Bi) Thiamin in the body is chiefly found in the phosphorylated form thiamin pyrophosphate (TPP) which is a coenzyme. The majority (80%) of thiamin in the blood is found in the erythrocytes and assay of blood thiamin is a more reliable indicator of deficiency than assay of erythrocyte transketolase. The phosphorylated vitamers are enzymically converted to thiamin in samples using diastase following deproteinization. To reach the low picomolar concentrations the thiamin compounds are oxidized by ferricyanide to form thiochromes, which are highly fluorescent. The thiochromes are then separated by reversed-phase HPLC and detected by their emission at 425-450 nm. 

In most organisms, the coenzyme ThDP is the most abundant thiamin compound. Free thiamin and ThMP generally amount to 5-15% of total thiamin. Thiamin is generally present in small amounts in E. coli it is hardly detectable (ThMP is the first thiamin derivative synthesized in this organism), while in mammalian tissues it may represent several percent of total thiamin. The tri-phosphorylated derivatives ThTP and AThTP are generally minor compounds in animal tissues, but they can be major thiamin compounds in E. coli under particular conditions of starvation (Bettendorlf et al. 2007). No data on the distribution of thiamin derivatives are available for bacteria other than E. coli. 

Cytosolic adenylate kinases may synthesize ThTP at a slow rate, both in prokaryotes and in animals. This mechanism is probably only relevant in tissues with high AK activity, such as skeletal muscle and electric organs. Therefore, in some species where, in addition to a high AK activity, soluble 25 kDa ThTPase is either absent or inactive (E. electric organ, pig and chicken skeletal muscle, see above), ThTP may accumulate in the cytosol, even becoming the major thiamin compound. 

Since thiamine does not fluorescence, the development of the thiochrome analysis or closely related fluorescent compounds has provided a highly specific method for thiamine quantification from most matrices. The development of HPLC methods, often coupled with either pre- or post-column oxidation to thiochrome, permits highly sensitive measurement of total thiamine or its individual free and phosphorylated forms (Gregory 1997). Thiochrome-formed pre-column refers to procedures that form the thiochrome directly in the sample extract. Post-column procedures are based on the conversion of thiamine compounds to their respective thiochromes after chromatographic resolution of the thiamine forms. The oxidizing agent is usually 0.01% potassium hexacyanoferrate(iii) [K3Fe(CN)6] solution in 15% sodium hydroxide. 

Paper partition chromatography (PPC) was the first used for separating thiamine phosphates in biological materials. A good PPC separation of thiamine phosphates was reported by using several different solvent systems. Photometry at 270 nm of eluted, individual spots allowed quantitation of each thiamine compound down to the lO-pg level. 

The detection of thiamine compounds in the eluate is carried out either spectro-photometrically, usually at 254 nm, or fluorometrically. 

In fluorometric detection, two different procedures are used. First, samples containing thiamine compounds are converted to thiochrome compounds using reagents for alkaline oxidation and then chromatographed (precolumn derivatiza-tion). In the second procedure, samples are first directly chromatographed, and the... 

In our HPLC system, thiamine compounds were converted to thiochromes by alkaline BrCN oxidation prior to separation. An ODS column was used as the stationary phase and 2.5% /V,/V-dimethylformamide (DMF)-25 mM potassium phosphate buffer (pH 8.4) as the mobile phase. The thiochromes were detected fluorometrically. After completion of ThcMP elution, the mobile phase was... 

L Bettendorff, P Wins, E Schoffeniels. Regulation of ion uptake in membrane vesicles from rat brain by thiamine compounds. Biochem Biophys Res Commun 171 1137-1144, 1990. 

Kawasaki, C., Modified thiamine compounds, in Vitamins and Hormones, Vol. XXI, Harris, R.S., Ed., Academic Press, New York, 1963. 

Thiamin Compounds Pyridine Compounds Imidazole Compounds Oxygen Compounds Creatine, Creatinine Polyamines... 

Beyond pharmaceutical screening activity developed on aminothiazoles derivatives, some studies at the molecular level were performed. Thus 2-aminothiazole was shown to inhibit thiamine biosynthesis (941). Nrridazole (419) affects iron metabohsm (850). The dehydrase for 5-aminolevulinic acid of mouse liver is inhibited by 2-amino-4-(iS-hydroxy-ethyl)thiazole (420) (942) (Scheme 239). l-Phenyl-3-(2-thiazolyl)thiourea (421) is a dopamine fS-hydroxylase inhibitor (943). Compound 422 inhibits the enzyme activity of 3, 5 -nucleotide phosphodiesterase (944). The oxalate salt of 423, an analog of levamisole 424 (945) (Scheme 240),... 

Thiazolium derivatives unsubstituted at the 2-position (35) are potentially interesting precursors of A-4-thiazoline-2-thiones and A-4-thiazoline-2-ones. Compound 35 in basic medium undergoes proton abstraction leading to the very active nucleophilic species 36a and 36b (Scheme 16) (43-46). Special interest has been focused upon the reactivity of 36a and 36b because they are considered as the reactive species of the thiamine action in some biochemical reaction, and as catalysts for several condensation reactions (47-50). 

The thiazole ring can be found in numerous molecules that possess biological activity the thiamine (vitamin B,), penicillins, antiinflamatory and bactericidals compounds, and so forth. 

Sulfur Dioxide and Sulfites. Sulfur dioxide [7446-09-5], SO2, sodium bisulfite [15181-46-1], NaHSO, and sodium metabisulfite [23134-05-6] ate effective against molds, bacteria, and certain strains of yeast. The wine industry represents the largest user of sulfites, because the compounds do not affect the yeast needed for fermentation. Other appHcations include dehydrated fmits and vegetables, fmit juices, symps and concentrates, and fresh shrimp (79). Sulfites ate destmctive to thiamin, and cannot be used in foods, such as certain baked goods, that ate important sources of this vitamin. 

Naturally occurring quaternary ammonium compounds have been reviewed (179). Many types of aliphatic, heterocycHc, and aromatic derived quaternary ammonium compounds are produced both in plants and invertebrates. Examples include thiamine (vitamin B ) (4) (see Vitamins) choline (qv) [62-49-7] (5) and acetylcholine (6). These have numerous biochemical functions. Several quaternaries are precursors for active metaboUtes. 

The yellow form (11) on acidification is converted to the more stable thiol form (12). On oxidation, typically with alkaline ferhcyanide, yellow form (11) is irreversibly converted to thiochrome [299-35-4] (14), a yellow crystalline compound found naturally in yeast but with no thiamine activity. In solution, thiochrome exhibits an intense blue fluorescence, a property used for the quantitative determination of thiamine. 

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

Thiamine is present in cells as the free form 1, as the diphosphate 2, and as the diphosphate of the hydroxyethyl derivative 3 (Scheme 1) in variable ratio. The component heterocyclic moieties, 4-amino-5-hydroxymethyl-2-methylpyrimidine (4) and 4-methyl-5-(2-hydroxyethyl)thiazole (5) are also presented in Scheme 1, with the atom numbering. This numbering follows the rules of nomenclature of heterocyclic compounds for the ring atoms, and is arbitrary for the substituents. To avoid the use of acronyms, compound 5 is termed as the thiazole of thiamine or more simply the thiazole. This does not raise any ambiguity because unsubstituted thiazole is encountered in this chapter. Other thiazoles are named after the rules of heterocyclic nomenclature. Pyrimidine 4 is called pyramine, a well established name in the field. A detailed account of the present status of knowledge on the biosynthesis of thiamine diphosphate from its heterocyclic moieties can be found in a review by the authors.1 This report provides only the minimal information necessary for understanding the main part of this chapter (Scheme 2). 

The thiazolecarboxylic acid structure (40) was also guessed in a similar way, from tracer experiments. The unknown compound was converted into the thiamine thiazole by heating at 100°C and pH 2. On paper electrophoresis, it migrated as an anion at pH 4. Tracer experiments indicated that it incorporated C-l and C-2 of L-tyrosine, and the sulfur of sulfate. The synthetic acid was prepared by carboxylation of the lithium derivative of the thiamine thiazole, and the derivatives shown in Scheme 19 were obtained by conventional methods. Again, the radioactivity of the unknown, labeled with 35S could not be separated from structure 40, added as carrier, and the molar radioactivity remained constant through several recrystallizations and the derivatizations of Scheme 17. 

Vitamins such as thiamin, biotin, and vitamin Bj2 are often added. Once again, the requirements of anaerobes are somewhat greater, and a more extensive range of vitamins that includes pantothenate, folate, and nicotinate is generally employed. In some cases, additions of low concentrations of peptones, yeast extract, casamino acids or rumen fluid may be used, though in higher concentrations, metabolic ambiguities may be introduced since these compounds may serve as additional carbon sources. 

Thiamin (vitamin B-l, 177) when photolysed, gives preparations having a characteristic odour. Photolysis of an aqueous solution with a high-pressure mercury lamp is reported to give the pyrimidine (178) [ 113]. Other work used irradiation at 254 nm and concentrated on the approximately 0.1% yield of ether-soluble odoriferous products. As many as nine compounds have been identified (179), (180), (181), 2-methyl-3-formyl-4,5-dihydrofuran, 3-acetyl-4,5-dihydrofuran, 4-oxopentyl formate, 3-formyl-5-hydroxypentan-2-one, 3-mercapto-2-methyl-4,5-dihydrofuran and bis(4,5-dihydro-2-methylfuran-3-yl)disulphide [114, 115]. 

The types of compounds that can be analyzed by fluorometry are rather limited. Benzene ring systems, such as the vitamins riboflavin (Figure 8.13) and thiamine, are especially highly fluorescent compounds and are analyzed in foods and pharmaceutical preparations by fluorometry. Metals can be analyzed by fluorometry if they are able to form complex ions by reaction with a ligand having a benzene ring system. 

The infrared technique has been described in numerous publications and recent reviews were published by Davies and Giangiacomo (2000), Ismail et al. (1997) and Wetzel (1998). Very few applications have been described for analysis of additives in food products. One interesting application is for controlling vitamin concentrations in vitamin premixes used for fortification of food products by attenuated total reflectance (ATR) accessory with Fourier transform infrared (FTIR) (Wojciechowski et al., 1998). Four vitamins were analysed - Bi (thiamin), B2 (riboflavin), B6 (vitamin B6 compounds) and Niacin (nicotinic acid) - in about 10 minutes. The partial least squares technique was used for calibration of the equipment. The precision of measurements was in the range 4-8%, similar to those obtained for the four vitamins by the reference HPLC method. 

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