Thiochrome Fluorescence Research Articles

Fluorometric Detection of Thiamine Based on Hemoglobin-Cu3(PO4)2 Nanoflowers (NFs) with Peroxidase Mimetic Activity.

Component analysis plays an important role in food production, pharmaceutics and agriculture. Nanozymes have attracted wide attention in analytical applications for their enzyme-like properties. In this work, a fluorometric method is described for the determination of thiamine (TH) (vitamin B1) based on hemoglobin–Cu3(PO4)2 nanoflowers (Hb–Cu3(PO4)2 NFs) with peroxidase-like properties. The Hb–Cu3(PO4)2 NFs catalyzed the decomposition of H2O2 into ·OH radicals in an alkaline solution that could efficiently react with nonfluorescent thiamine to fluoresce thiochrome. The fluorescence of thiochrome was further enhanced with a nonionic surfactant, Tween 80. Under optimal reaction conditions, the linear range for thiamine was from 5 × 10−8 to 5 × 10−5 mol/L. The correlation coefficient for the calibration curve and the limit of detection (LOD) were 0.9972 and 4.8 × 10−8 mol/L, respectively. The other vitamins did not bring about any obvious changes in fluorescence. The developed method based on hybrid nanoflowers is specific, pragmatically simple and sensitive, and has potential for application in thiamine detection.

A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates

A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C18 column using a mixture of 50mmolL−1 borate buffer pH 9.0 containing 0.25mmolL−1 tetrabutylammonium chloride and 0.5mmolL−1 EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440nm with excitation at 375nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmolL−1 level), accurate, specific, rapid (analysis time approximately 13min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05molL−1 ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid.

A Rapid Solid‐Phase Extraction Fluorometric Method for Thiamine and Riboflavin in Salmonid Eggs

AbstractA new method has been developed and successfully applied to the selective measurement of thiamine (nonphosphorylated), total thiamine (sum of thiamine, thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP)), and potentially interfering riboflavin in acidic (2% trichloroacetic acid) extracts of selected salmonid and walleye egg samples. Acidic extracts of eggs were applied directly to end‐capped C18, reversed‐phase solid‐phase extraction (SPE) columns and separated into three fractions by elution with mixtures of PO4 buffer (pH 2), methanol (10%), and acetonitrile (20%). All thiamine compounds recovered in the first two fractions were oxidized to their corresponding thiochromes with alkaline potassium hexacyanoferrate, and we measured the thiochrome fluorescence (excitation at 360 nm, emission at 460 nm) in a 96‐well microplate reader. Riboflavin, recovered in third fraction (eluted with pH 2, 20% acetonitrile), was analyzed directly by measuring the fluorescence of this fraction (excitation at 450 nm, emission at 530 nm). Significant portions of the phosphate esters of thiamine (TMP, TDP, and presumably TTP), when present at low concentrations (<10 nmol of total −thiamine per gram of egg), were not retained by the 100‐mg SPE column, and were collected directly during sample loading and in a subsequent phosphoric acid rinse as fraction 1. Free thiamine (nonphosphorylated) and remaining portions of the TDP and TMP were then eluted in the second fraction with 10% methanol/PO4 buffer, whereas the un‐ionized, relatively nonpolar riboflavin was eluted in the third fraction with 20% acetonitrile. This new method uses a traditional sample homogenization of egg tissue to extract thiamine compounds into 2% trichlororacetic acid solution; an inexpensive, commercially available SPE column; small amounts of sample (0.5–1 g); microliter volumes of solvents per sample; a traditional, relatively nonhazardous, oxidation of thiamine compounds to fluorescent thiochromes; and an ultraviolet–visible‐wavelength‐filter fluorometer for the measurements.

The Changes in Vitamins Content in Foods Soaked in the Chlorine Dioxide Solution.

Twelve sorts of foods were tested to investigate the changes in the contents of ascorbic acid, vitamin B1 and vitamin B2 by soaking in 0.5 ppm chlorine dioxide solution. These vitamins in food were extracted with ultrasonic generator for 30 min, and the percentages of the loss of ascorbic acid, vitamin B1 and vitamin B2 were determined by the dichlorphenolindophenol method, thiochrome fluorescence method and lumifulavin method, respectively. It was found that there were the loss of 0-5% in the contents of vitamins after soaking for 15 min and 15-30% after 60 min. However, the percentage of the loss induced by the chlorine dioxide solution was less than that induced by 0.5 ppm chlorite, 0.5 ppm hypochlorite and potable water. The existence of Fe3+, Cu2+ in the solution had an obvious effect which accelerates the decomposition of ascorbic acid.

Fluorescence high-performance liquid chromatography of trace microamounts of phosphate by oxidation of thiamine to thiochrome

A highly sensitive fluorescence high-performance liquid chromatography, based on the fluorescence of thiochrome which is formed from oxidation of thiamine by the molybdoheteropoly acid of phosphate has been developed for the determination of trace microamounts of phosphate. The method permits the determination of phosphate in the range of 0.1 to 1.0 ng as P/10 Μl sample solution (3.23×10−7 to 3.23×10−6 mol l−3 as P or PO4), and it may be possible to apply the proposed method to the microanalysis of phosphate in various biological samples.

Comparison of Manual and Benzenesulfonyl Chloride-Semiautomated Thiochrome Methods for Determination of Thiamine in Foods

A semiautomated procedure was used to measure the fluorescence of sample extracts before and after the addition of benzenesulfonyl chloride (BSC). Addition of BSC inhibited thiochrome formation and provided a more representative blank based on the fluorescence of all the reactants except thiochrome. Thiamine standard was added to each sample extract so that thiamine concentration could be calculated after correcting for sample matrix effects on thiochrome fluorescence. Twenty food products were analyzed using this method, and the results were compared with those obtained using the manual AOAC method. The mean percent recoveries and standard deviations were 100.2 +/- 5.3 and 101.1 +/- 10.1 for the BSC-semiautomated and the AOAC manual methods, respectively. Replicate analyses using the BSC method gave an average coefficient of variation of 2.8%. Linear regression analysis showed that the BSC method gave higher values, with a mean increase of 14.8%, than those obtained using the manual method. Sixty-four percent of this difference was due to elimination of the column purification step and 36% was due to correcting for sample matrix effects on thiochrome and blank fluorescence. The BSC method provides a rapid, accurate, and reproducible method for thiamine assay in different food products, especially for those foods with low thiamine levels.

Zur quantitativen Vitamin-B1-Bestimmung in Nahrungsmitteln und biologischem Material

A fluorometric method for the estimation of the vitamin B1 content in food and biological materials is described. Acid hydrolysis is followed by enzymatic treatment to liberate the vitamin B1 of the sample. Interfering substances in the extract are removed to a great extent by chromatography on Amberlite CG 50, a weakly acidic cation exchanger. After oxidation and extraction into an organic medium, the fluorescence of thiochrome is measured. The procedure is simple, reproducible and not susceptible to interference. The recoveries are good.

Thiamine Whole Blood Pharmacokinetics in Rats Using Both a Specific 35S-Thiamine Liquid Scintillation Assay and the Thiochrome Fluorescence Assay

The study of factors altering the CNS and GI absorption of thiamine. In rats required the development of a specific assay for thiamine from 100-μl samples of blood and plasma and small quantities of tissue. The specific thiochrome fluorescence assay for thiamine was modified to handle microsamples and to use 35S-thiamine. This sensitive and specific radioassay using 35S-thiamine gave pharmacokinetic parameters for 4-mg/kg iv doses of thiamine in rats equivalent to those using the less sensitive thiochrome fluorescence assay. The new assay, because of its lower limit of detection, allowed the study of the time profile of thiamine after a 1-mg/kg iv dose in rats. Such a time profile could not have been followed using the standard thiochrome fluorescence assay.

A fluorometric assay of proteins on Kieselguhr plates using sodium hypochlorite and thiamine

A simple, rapid, and sensitive method for the fluorometric assay of proteins is described in this communication. Proteins are spotted on a Kieselguhr G plate and N-chlorinated by spraying with a solution of sodium hypochlorite. After removal of excess hypochlorite by aeration, the plate is sprayed with a solution of thiamine. The fluorescence of thiochrome, formed by the oxidation of thiamine with the N-chlorinated proteins, is then measured with a scanning fluorometer. Interfering substances including DNA are readily removed by the development of the plate with 2 m sodium chloride prior to the assay. The standard curves were linear in the range of 0.1 to 1.0 μg/spot of proteins, and the fluorescence intensity deviated only slightly among proteins.

Differences in Thiochrome Fluorescence Produced by Thiamine and Its Mono-, Di-, and Triphosphate Esters.

Differences in Thiochrome Fluorescence Produced by Thiamine and Its Mono-, Di-, and Triphosphate Esters.

A simple automated method for the determination of urinary thiamin

An automated method for measuring urinary thiamin without isolating the thiamin is described. Two ahiquots of sample are treated alike prior to measurement of thiochrome fluorescence, except that in the blank, addition of benzene-sulfonyl chloride and sodium hydroxide blocks oxidation of thiamin to thiochrome. Thus, fluorescence due to nonspecific substances can be subtracted. Quenching is negligible.

Coenzyme binding in yeast pyruvate decarboxylase. A fluorescent enzyme-inhibitor complex.

The formation of a fluorescent complex between apopyruvate decarboxylase of Saccharomyces carlsbergensis and thiochrome pyrophosphate, a competitive inhibitor with thiamine pyrophosphate, and Mg(II) ions is reported. A similar complex between thiochrome and the apoenzyme could not be detected, demonstrating the importance of the pyrophosphate group of the coenzyme in binding to the protein. Complex formation between the apoenzyme and thiochrome pyrophosphate in the absence of Mg(II) could also not be demonstrated. The role of magnesium in coenzyme binding is discussed, especially in reference to interaction with the pyrophosphate moiety. The apparent Km for Mg(II) ions, after correction for residual apoenzyme activity, is reported to be 87 μM.The shift in the fluorescence emission spectrum of thiochrome pyrophosphate of 23–24 nm toward lower wavelengths, upon complex formation, coincided with the behavior of thiochrome in solvents of lower polarity than water. A hydrophobic region at the coenzyme binding site of pyruvate decarboxylase is proposed. Enhancement of thiochrome fluorescence occurred concomitantly with the blue shift in the emission spectrum upon complex formation.The excitation wavelength of 290 nm employed to demonstrate complex formation suggested energy transfer from the tryptophan to the thiochrome chromophore. Quenching of fluorescence in the 340–350 nm region, however, occurred not only in the protein emission spectrum upon complex formation, but also when free tryptophan and thiochrome interacted with each other. The quenching of the protein fluorescence appears to be independent of hydrophobic interaction at the coenzyme binding site. It is suggested that protein tryptophan residues relatively exposed to solvent interact individually with the thiochrome molecule by an energy transfer mechanism to produce the quenching in pyruvate decarboxylase.

Cytokinin Activation of de novo Thiamine Biosynthesis in Tobacco Callus Cultures

The effect of kinetin on the de novo formation of thiamine in tobacco callus cultures was measured by following the isotope dilution of previously introduced (14)C-thiamine. Thiamine was determined by the thiochrome fluorescence assay after chromatographic purification. Morphological effects induced by high kinetin concentrations were visible within a week after tissue transfer, but thiamine synthesis was insignificant for 2 weeks both in cultures with high (1000 mug/l) and low (30 mug/l) kinetin treatments. Thiamine synthesis during the third week was observed at both kinetin levels, the high kinetin treatment supporting 2.5 times the thiamine synthesis of the low kinetin treatment. The kinetin induced increases in thiamine observed earlier by Digby and Skoog apparently resulted from stimulation of thiamine synthesis rather than from sparing its destruction. Thiamine synthesis is initiated when thiamine concentration reaches a minimum in the callus tissue. This suggests that kinetin is required for the synthesis, but that the activation of synthesis is under feedback control sensitive to the level of thiamine in the tissue.

The fluorescent microscopic localization of thiamine in nervous tissue.

A new fluorescent microscopic technique for visualizing thiamine in nervous tissue is described. Freeze dried sections are exposed to the vapors of cyanogen bromide and ammonia to convert thiamine to its fluorescent derivative thiochrome. The specificity of the procedure is ensured by utilizing monochromatic light for activation and a narrow band filter for emission and by scanning fluorescence after exposing the preparation to ultraviolet light at 365 mµ to destroy the thiochrome fluorescence. With this technique it has been shown that, with every preparation of nervous tissue that has been examined, thiamine is present only in membranes and not in axoplasm. This finding is discussed in relation to a suggested role of the vitamin in ion transport.

Cytokinin Activation of Thiamine Biosynthesis in Tobacco Callus Cultures

Bioassays of tissue extracts show that high (500-1000 mug/liter) kinetin concentrations which permit growth of tobacco callus cultures on media without added thiamine activate the biosynthesis of this vitamin by the tissues. Although the tissue concentration of thiamine may fall appreciably, it is maintained at a level adequate for survival and slow growth of the cultures, and there is a large net increase in total thiamine content per culture with time. In the second and subsequent passages of tissue on a thiamine free medium, growth is obtained only when high kinetin concentrations are maintained. Effective inhibition of growth by antithiamines suggests that thiamine is utilized by the high-kinetin tissue.In the presence of low (30-100 mug/liter) kinetin concentrations, which would be optimal for growth in the presence of thiamine, growth only occurs early in the first passage of tissue from a medium with the vitamin to one without it. The thiamine concentration in the tissues falls to low levels, and no net biosynthesis is apparent. The tissues turn dark and die after 2 to 3 weeks. In contrast with this, in the absence of both added thiamine and kinetin no appreciable growth occurs, but the tissues keep their normal appearance, retain their thiamine content, and may stay alive for several weeks.

Simultaneous determination of thiamine and pyrithiamine

1. (1) Alkaline potassium ferricyanide oxidation of thiamine and pyrithiamine yields two different products as judged by excitation and emission characteristics. The former oxidation produces thiochrome and the latter pyrichrome. 2. (2) The differences in spectral characteristics of thiochrome and pyrichrome have been utilized to determine quantitatively thiamine and pyrithiamine concentration in a mixture of the two. 3. (3) Two generalized equations were derived to determine the concentration of thiamine and pyrithiamine in the presence of biological material: T = d ad − bc E 435 385 − b ad − bc E 480 385 = mνmoles thiamine/asay P = a ad − bc E 480 385 − c ad − bc E 480 385 = mνmoles pyrithiamine/asay where E = emission, P = pyrithiamine, T = thiamine, the numerator of the subscripts the emission wavelength, and the denominator the excitation wavelength. Constants a and b represent the relative molar fluorescence of thiochrome and pyrichrome when excited at 385 mμ and emitting at 435 mμ. Constants c and d represent the relative molar fluorescence of the same two respective compounds with the emission measured at 480 mμ and excitation at 385 mμ. 4. (4) The limit of detection for both thiamine and pyrithiamine is determined both by their absolute amounts and by the molar ratio of the two. When the molar ratio of pyrithiamine to thiamine was between 40 and 8, 12 mμmoles of pyrithiamine could be detected with recoveries ranging from 90 to 115%. With a similar range of molar ratios, 1.5 mμmoles of thiamine per assay could be detected with recoveries ranging from 90 to 100%. The limitations of this approach have been considered.

穀菽類のビタミンB<sub>1</sub>の螢光写真による研究

(1) Studies have been made about the distribution of thiamine in the seed of kidney bean at different stages of germination (kept at 25°, in the dark, for 14 days) by the photographic method of thiochrome fluorescence, and in each stage the moisture content, fresh-weight of one plant and the thiamine content (both total and free) were measured. (2) In regard to the thiamine contents in each fraction of the germinated seed, it was found to decrease after 2 days' germination in the cotyledon, but to increased rather rapidly in embryo. The whole thiamine contents of one seed decrerased after 2 days' germination. This is supposed to be the reason, why synthesis of thiamine did not occur during the dark germination, only consumption proceeding, (3) In regard to the distribution of thiamine fluorescence, there was nothing notable concerning this matter as no marked change was observed in each fraction of seed through 14 days' germination, but when the lateral root developed, strong fluorescence concentrated in the primordium and the procanbium of the transitional region. This strong fluorescence was recognized in the fraction which developed vigorously.

Studies on thiamine in cereals by a photographic method. I. The distribution of thiamine in domestic rice at different stages of maturity.

The distribution of thiamine in the domestic wheat, barley and naked barley at different stages of maturity was studied by the photographic method of thiochrome fluorescence, and moisture content, weight per thousand kernels and thiamine content were measured. Moreover, the distribution of thiamine in each mature kernel was studied in detail.

Studies on the distribution of thiamine in the seed and seedling of kidney bean by the photographic method.

The distribution of thiamine both in the dormant seed and in the seedling of kidney beans at different stages of germination (kept at 25°, for 14 days in the dark) was studied by the photographic method of thiochrome fluorescence and the moisture content, fresh weight per plant and thiamine content (total and free) of each stage were measured.

STUDIES ON THIAMINE IN CEREALS BY A PHOTOGRAPHIC METHOD

The distribution of thiamine in the domestic wheat, barley and naked barley at different stages of maturity was studied by the photographic method of thiochrome fluorescence, and moisture content, weight per thousand kernels and thiamine content were measured. Moreover, the distribution of thiamine in each mature kernel was studied in detail.