Thiochrome Method Research Articles

Перспективы повышения витаминной ценности пищевых концентратов – бульонных кубиков

Introduction. The lack of micronutrients in the diet of the Russian population continues to arouse the interest of the scientific community in the development of fortified foods. The range of enriched products on the domestic market remains quite poor. Bouillon cubes are popular in home cooking and catering. The data of the scientific and technical literature indicate an almost complete absence of micronutrients, e.g. vitamins, minerals, and trace elements, in bouillon cubes, with the exception of iodine when the formulation included iodized salt. There have also been successful attempts to fortify bouillon cubes with iron. Taking into consideration the high level of vitamin B deficiency in Russian population, the research objective was to assess the possibility of fortification of dry food concentrates to obtain bouillons rich in vitamins and iron. Study objects and methods. The research featured dry food concentrate “Dry bouillon with chicken flavor” and the vitamin and mineral premix “Kolos Forte” containing five B vitamins and iron in the form of sulfate. The content of vitamin B1 was determined fluorometrically by thiochrome method, and vitamin B2 was determined by fluorometric titration with riboflavin-binding protein. Results and discussion. The premix was selected based on a thorough analysis and systematization of the material on the research topic. The addition of a vitamin-mineral premix to the food concentrate did not affect the sensory properties of both dry concentrate and reconstituted bouillon. The added vitamins proved highly stable, amounting to 95–100%. The vitamin-mineral premix containing B vitamins and iron made it possible to increase the content of these micronutrients in a 200 mL bouillon portion to 19–30% of the recommended daily intake. Conclusion. The diet of different population groups still remains poor in micronutrients, which makes food fortification an urgent task. The present article offers a comparative analysis of the nutritional value of chicken broths and those obtained from cubes fortified with vitamin and mineral premix. The vitamin value of one portion of reconstituted broth fortified with a vitamin and mineral premix which contained 19–30% of the recommended daily intake of vitamins and iron proved 2–5 times higher than that of actual meat broths. The research resulted in a new formulation for bouillon cubes with a vitamin-mineral premix containing five B vitamins and iron. The increased micronutrient value of the developed fortified product made it possible to recommend it as an additional source of vitamins and iron.

Nutrient value of fruit in promising strawberry varieties

One of the current areas in research of fruit and berry crops is the study of their nutritional value and micronutrient composition. Valuable chemical composition of the strawberry determines a wide spectrum of its utilization not only as fresh fruit, but also as a raw material for food industry. Breeding requirements for contemporary strawberry varieties together with yield, large-fruit, important taste and dietary qualities of fruit provide for higher content of biologic active substances. The aim of the research was an integrated assessment of strawberry fruit for content of nutrients and bioactive compounds, as well as mineral elements, and its antioxidant activity. Material and methods. The objects of investigation were berries of 5 strawberry varieties bred in the I.V. Michurin Scientific Centre (Lakomaya, Prazdnichnaya, Privlekatelnaya, Urozhaynaya CGL, Feyerverk) and 9 foreign varieties (Vima Zanta, Dukat, Zefir, Kama, Maryshka, Red Gauntlet, Festivalnaya romashka, Honey, Elsanta) which are promising for cultivation in the Black Soil Region of Russia. The content of soluble solids was determined by the refractometric method, sugars - by Bertrand's method, pectins - by the volumetric method, anthocyanins - by pH-differential spectrophotometry. Total content of antioxidants was defined by amperometric method with recalculation per quercetin. Determination of vitamin B1 was carried out using the fluorometric thiochrome method, vitamin B2 - by fluorometric titration with riboflavin-binding apoprotein after acid-enzymatic hydrolysis, ascorbic acid, vitamin E (tocopherols), niacin, organic and hydroxycinnamic acids - by HPLC, flavonols - spectrophotometrically, minerals and trace elements - by atomic absorption method. Results and discussion. A significant variation in the chemical composition indices depending on the varietal characteristics was found. Strawberry fruit accumulated 8.5-12.0% soluble solids, 5.9-8.7% sugars, 1.5-2.1% of fibre, 0.78-1.12% pectin substances, 0.89-1.45% organic acid; 100 g contain 33.5-48.2 mg vitamin C, 18.3-108.5 mg anthocyanins, 0.024-0.041 mg vitamin B1, 0.012-0.029 mg vitamin B2, 0.10-0.26 mg niacin, 0.26-0.60 mg TE vitamin E, 102-270 mg potassium, 0.15-0.95 mg iron; total antioxidant activity ranged within 180.8-350.0 mg/100 g. High level of anthocyan accumulation (over 80.0 mg/100 g) was observed in Privlekatelnaya, Lakomaya and Feyerverk strawberry varieties. Urozhaynaya CGL, Privlekatelnaya and Lakomaya varieties were characterized by dessert taste. According to the results of the studies Privlekatelnaya variety stood out because of the high complex accumulation of vitamin C, anthocyanins, and high total antioxidant activity. These varieties are recommended for both fresh use and for freezing, as well as for production of functional foods. Conclusion. The obtained data are of interest for further inclusion in the Tables of the chemical composition of Russian food products.

Flow-injection solvent extraction without phase separation: Fluorimetric determination of thiamine by the thiochrome method

Two modes of liquid–liquid extraction in flow-injection systems were compared and applied to the fluorimetric determination of thiamine (Vitamin B 1). The first included phase segmentation, but fluorescence was measured without phase separation. In this mode, thiamine was detected at concentrations higher than 8 μg/l with a linear application range of 30–2000 μg/l, an R.S.D. of 1.9% (150 μg/l, n = 10) and a sampling frequency of 60/h. In the second mode, a single segment of organic solution was injected into the aqueous stream and fluorescence was also measured without phase separation. Using this mode, concentrations of thiamine higher than 1 μg/l were detected, with a linear application range between 5 and 280 μg/l, an R.S.D. of 2.4% (150 μg/l, n = 10) and a sampling frequency of 60/h. The two forms were applied to the analysis of thiamine in pharmaceuticals.

Indirect spectrophotometric determination of thiamine in pharmaceutical preparations.

An indirect, sensitive spectrophotometric method for the assay of thiamine is described. The procedure is based on the formation of a mercaptide by thiamine, with a known excess of silver ions in a buffered medium of pH 9.0 +/- 0.1. The unreacted silver ions are determined by the formation of an ion-pair complex with 1,10-phenanthroline and 2,4,5,7-tetrabromofluorescein, which shows an absorption maximum at 550 nm. The absorbance is found to decrease linearly with increasing concentrations of thiamine, which is corroborated by the calculated correlation coefficient value of -0.998. The system obeys Beer's law for 0-25 micrograms of thiamine in an overall aqueous volume of 10 ml. The molar absorptivity and relative standard deviation (RSD) were calculated to be 7.8 x 10(4) l mol-1 cm-1 and 2.5% (n = 10), respectively. The proposed method was applied successfully to the determination of thiamine in pharmaceutical preparations. The reliability of the assay was established by parallel determination by the standard thiochrome method and recovery studies.

8] Determination of thiamin and its phosphate esters in human blood, plasma, and urine

This chapter focuses on the determination of thiamin and its phosphate esters in human blood, plasma, and urine. There are several functional tests for the estimation of triphosphate (TPP) concentration. Among them are the glucose tolerance test and the erythrocyte transketolase assay. The transketolase assay has become a standard test used to estimate thiamin deficiency. The thiochrome method introduces the oxidation of thiamin to the fluorescent thiochrome, which is then determined by fluorometry. The use of high-performance liquid chromatography (HPLC) allows separation of the thiamin compounds under analysis. The chapter proposes a new method, which combines the advantages of high sensitivity and determination of the four compounds in one chromatographic run. The conversion of thiamin compounds into fluorescent derivatives is obtained by alkaline oxidation, which converts thiamin into the fluorescent thiochrome. Cyanogen bromide is chosen as it is the most efficient oxidizing agent.

11] Cyanogen bromide-based assay of thiamin

This chapter describes the thiochrome method used to develop a low-cost thiamin assay sufficiently sensitive for pediatric application in both whole blood and cerebral spinal fluid, and the normative ranges for those biological fluids. It presents reagents used and procedure employed for sample preparation. For whole blood, the within-run coefficients of variations (CVs) are 3.6% (total) and 4.6% (nonphosphorylated). The long-term between-run CVs (control blood over 40 weekly assays) are 6.0% (total) and 15.5% (nonphosphorylated). The total and nonphosphorylated thiamin values are moderately higher in blood samples from heparinized vs EDTA tubes. There is a strong negative correlation for both total and phosphorylated thiamin with age. The greatest decline occurs in the first three months of age, with a smaller additional decrease from 3–12 months. Thereafter, thiamin levels are comparable to adults. The decline occurs relatively independently of the changes in hematocrit characteristically seen in infancy. There are no differences by sex at any age or by race if less than 10 years of age.

Determination of thiamine in dried yeast by high-performance liquid chromatography using a clean-up column of CM-cellulose

Abstract Thiamine in dried yeast was cleaned up with a CM-cellulose column and determined by HPLC on a reversedphase ODS column with sodium 1-octanesulfonate as an ion-pairing agent and UV detection. Thiamine and phenacetin as an internal standard were monitored with UV detection at 254 nm. The calibration graph was linear in the range 0.25–4 μ g/ml of thiamine (as thiamine hydrochloride) with a correlation coefficient of 0.998. Intra- and inter-day variations of thiamine in dried yeast were 6.5 and 6.9%, respectively. Thiamine was recovered in good yield (97.8–105.7%, n = 5). By using this method, the thiamine content in dried yeast was found to be 138–169 μ g/g. These results were in good agreement with those of the conventional thiochrome method.

Analysis of thiamine in dried yeast by high-performance liquid chromatography and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry

High-performance liquid chromatography (HPLC) and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) have been applied to the analysis of thiamine in dried yeast. Thiamine was extracted from dried yeast with isobutanol containing sodium 1-octanesulfonate as an ion-pairing agent and determined by HPLC on a reversed phase ODS column with UV detection at 254 nm. Response was linear in the range 25–300 μg/g of thiamine in dried yeast with a coefficient of variation in the reproducibility of 8.0%. Thiamine was recovered in good yield (109.2%, n=5). Identification of the thiamine peak was obtained by the mass spectrum using the HPLC/APCI-MS system. The utility of the selected ion monitoring technique using the HPLC/APCI-MS was also investigated. The results obtained by this method are in good agreement with those obtained by the thiochrome method [1].

Automated microbiological assay of thiamin in serum and red cells.

To develop a sensitive, direct, automated method for the measurement of serum and red cell thiamin. A microbiological assay using a chloramphenicol resistant strain of Lactobacillus fermenti as the test organism was developed. Addition of chloramphenicol and cycloheximide to the assay medium suppressed bacterial and yeast contamination and enabled tests to be automated without recourse to aseptic procedures. Evaluation of the assay included precision analysis and estimation of thiamin recovery. Results obtained on red cell extracts were compared with an established colorimetric (thiochrome) method. Acceptable intrabatch and interbatch precision was obtained and good recovery of thiamin added to serum was obtained. Non-parametric reference ranges based on the results from 505 healthy people were: serum thiamin 11.3-35.0 nmol/l and red cell thiamin 190-400 nmol/l. Results were not age or gender related. The method gave results for red cell thiamin which were significantly higher than those obtained with an established thiochrome method. This automated microbiological assay is sensitive to 2.0 nmol/l of thiamin and allows tests to be set up at the rate of 100 per hour and after 20-22 hours allows incubation results to be read at 60 per hour. The method has proved reliable, suitable for the assay of large numbers of samples, and relatively inexpensive to perform.

Competitive protein-binding radioassay of thiamine in selected biological materials

A principle of competitive protein-binding radioassay is developed for thiamine determination in some biological samples. Thiamine in an assay sample competes with radiolabelled thiamine for Sepharose-immobilized buckwheat-seed thiamine-binding protein. A blank sample is prepared by destruction of theiamine in hot alkaline solution. Model studies show that the radioassay works in thiamine concentration range of 1–10 μM, in samples of moderate ionic strenght (up to 0.25 M NaCl) and is specific for thiamine in the presence of up tp 5-fold molar excess of thiamine phosphates. Thiamina phosphates can be determined but after hydrolysis with a suitable phosphatase enzyme (Taka-Diastase). Using this method, thiamine contents are successfully determined: (i) in spinach juice, directly, (ii) in cow's milk, after deproteinization, and (iii) in human urine, after desalting. Both the precision (C.V. less than 15%) and the recovery of thiamine supplements (82–100%, depending on thiamine pre-extraction method) are reasonable. Results of thiamine radioassay show a good correlation with control determinations by the standard thiochrome method.

Determination of thiamine (vitamin B1) in maternal blood during normal pregnancies and pregnancies with intrauterine growth retardation

The concentration of thiamine (vitamin B1) was measured in blood cells and plasma from mothers with normal pregnancy and from mothers whose pregnancy was complicated by intrauterine growth retardation (IUGR). Thiamine concentrations were estimated by the thiochrome method using HPLC separation and fluorimetric detection according to Weber et al. (2). During normal pregnancies the thiamine values in blood cells fall in the 28th to the 39th week of gestation from 230 nmol/l to 170 nmol/l. In cases with severe IUGR there is only a slight decrease in the thiamine levels from 140 nmol/l in the 30th week of gestation to a level of 130 nmol/l in the 39th week of gestation. During this period the thiamine values in plasma remain constant. To compare the thiamine concentrations in normal pregnancies and in those with IUGR we separated the thiamine values in a group from 28/0 to 34/6 and one from 35/0 to 39/6 weeks of gestation. In both groups mothers with normal pregnancy had significantly higher thiamine levels in the blood cells than mothers whose pregnancy was complicated by IUGR (p = 0.0001 and p = 0.0005). However, the thiamine values in plasma were not significantly different in normal pregnancies and pregnancies with IUGR. The results indicate that maternal thiamine deficiency might be one cause of IUGR.

Factors affecting a cyanogen bromide-based assay of thiamin.

We analyzed extensively a modified thiochrome method for thiamin analysis. Acid phosphatase (EC 3.1.3.2) from potato was superior to either alpha-amylase or acid phosphatase from wheat germ as a dephosphorylating agent. Timing of cyanogen bromide exposure was important, but the assay had good precision and accuracy. The standard curve was linear from 10 to 3000 nmol/L. The within-run and between-run coefficients of variation for total thiamin in whole blood were 3.6% and 7.4%, respectively. Analytical recoveries for low, intermediate, and high additions of thiamin to whole blood were 93-109%. Sample yield was increased by 41% (+/- 29% SD) with pre-assay freezing. Samples were stable for two days at room temperature, for seven days when refrigerated, and for two years when frozen. Previously unreported interference was seen with penicillin derivatives, and with several commonly used diuretic and antiepileptic medications. This assay may be suitable for population screening; 200 samples could be analyzed weekly at a cost of +0.20 per sample.

Infantile beriberi presenting as subacute necrotizing encephalomyelopathy

A case report of a breast-fed infant with formula supplementation showed it to suffer from infantile beriberi with lesions similar to those observed in patients with subacute necrotizing encephalomyelopathy. A modified thiochrome method was used to measure thiamine content; lesions were revealed by a CT scan. Other cases of infantile beriberi reported in the U.S. since 1958 occurred in infants for whom an unusual formula had been prescribed; it would seem prudent that any restrictive diet should be administered with caution and supplemented with a multivitamin preparation containing thiamine

Effect of fermentation on the thiamin, riboflavin and niacin contents of melon seed ( Citrullus vulgaris) and African oil bean seed ( Pentaclethra macrophylla)

An automated thiochrome method and high performance liquid chromatography (HPLC) with fluorescence detection were used to determine thiamin and riboflavin, respectively, in unfermented, and fermented melon seeds ( Citrullus vulgaris) and African oil bean seeds ( Pentaclethra macrophylla). Niacin was determined microbiologically. In all the methods used, fermentation significantly increased the content of thiamin, riboflavin and niacin in African oil bean seeds and thiamin and riboflavin in melon seeds. There was no change in the niacin content of melon seeds.

A reliable semiautomated method for the determination of total thiamine in whole blood by the thiochrome method with high-performance liquid chromatography.

A sensitive and reliable method for the determination of total thiamine (vitamin B1) in whole blood has been developed which is suited for routine analysis. After extraction, and enzymatic hydrolysis of thiamine phosphate esters, thiamine is separated from interfering compounds by a fully automated high-performance liquid chromatographic (HPLC) system. Thiamine is detected fluorometrically as thiochrome. By calculating the concentration of thiamine on-line with the aid of a computer, it is possible to complete one analysis within four hours. Routine thiamine determinations can be carried out in a series of 120 samples within 48 hours. The within-assay and between-assay coefficients of variation of the analysis of total thiamine in whole blood were 4.2 and 4.4%, respectively. The between-assay analytical recovery of thiamine diphosphate added to blood samples was 99.9 +/- 11.7% (mean +/- SD). The HPLC method described has been applied also to the analysis of thiamine in plasma and erythrocytes. In agreement with other reports, it was found that about 80% of total thiamine of whole blood is present in the erythrocytes. Reference values of thiamine in whole blood of the human were found in the range 95-155 nmol/l.

Effect of Radiation and Conventional Processing on the Thiamin Content of Pork

ABSTRACTA study was undertaken to compare the effect of ionizing radiation and thermal processing on the thiamin content of pork. Ground pork was either thermally processed in cans or, after enzyme‐inactivation, irradiated in pouches with electrons or gamma rays. Thiamin in treated and untreated samples was measured by the thiochrome method. The results show that thiamin retention in pork decreases as the irradiation dose increases, but that retention increases as the temperature of irradiation decreases. Moreover, irradiation with gamma rays (low dose‐rate) leads to lower retention than irradiation with electrons (high dose‐rate). Results also show that conventional thermal processing causes loss of thiamin comparable to or greater than irradiation at effective processing conditions for sterilization.

Determination of urinary thiamin by the thiochrome method

A modified procedure for the determination of thiamin in human urine by the thiochrome fluorescent method was used to examine the effects of three oxidizing agents. Urinary thiamin values obtained with the use of HgCl2 as the oxidizing agent were lower than those found with the use of either BrCN or K3Fe(CN)6. This may be due to a difference in the reactivity of interfering compounds with the specific oxidizing agent used. Recovery values were acceptable for each of these three oxidizing agents, although BrCN appears to be slightly superior. In addition to the recovery values, the linear response for the thiamin standards used in this study supports the use of a smaller resin column for the pretreatment of urine samples; this was found to be more economical and convenient to use.

Extraction based on the flow-injection principle: Part 3. Fluorimetric Determination of Vitamin B 1 (Thiamine) by the Thiochrome Method

The thiochrome method for determination of vitamin B 1 in pharmaceutical preparations has been adapted to a continuous flow system based on the flow-injection principle. The sample volume required for an analysis is about 150 /sml. For routine purposes a concentration range of 3 × 10 -4–6 × 10 -4 mg ml -1 is used. Results obtained with the system agree well with results obtained manually. The consumption of organic phase is 2–3 ml/sample and the sampling rate is 30/h. A sampling rate of 70/h is easily attained if necessary. The relative standard deviation is about 1%.

Thiamin requirement of the adult human

Young adult male subjects maintained on a metabolic ward were fed diets providing controlled intakes of thiamin and either 2800 or 3600 kcal. The higher level of calories was attained by an increased intake of carbohydrates. Constant weights were maintained by the subjects by adjusting daily activity and exercise schedules. Thiamin requirements were evaluated in terms of erythrocyte transketolase activity and urinary excretion of the vitamin. The results of the study revealed that a relationship exists between thiamin requirement and caloric intake and expenditure. Thus, when the calories being utilized were derived primarily from carbohydrate sources, the minimum adult male requirement for thiamin appeared to be 0.30 mg of thiamin per 1000 kcal. Urinary excretion of thiamin and erythrocyte transketolase activity appear to be reasonably reliable reflections of thiamin intakes and thiamin nutritional status. The use of these measurements in nutrition surveys appears justified. The microbiological assay (Lactobacillus viridescens) for measuring thiamin levels in urine samples appears to be a somewhat more sensitive but valid procedure as an alternate for the thiochrome method. Judged from the results of this study, the recommended intake for the adult human of 0.40 mg of thiamin per 1000 kcal by FAO/WHO and the recommended allowance of 0.5 mg per 1000 kcal by the Food and Nutrition Board of the NAS-NRC appear reasonable and amply allow for biological variations and other factors that may influence the requirement for this vitamin.

An improved method for the determination of endogenous thiamine and its phosphate esters in biological material.

Nonphosphorylated thiamine was extracted from tissue homogenates in saline or sucrose into ethylbutylketone-acetonitrile using counter-ion extraction and reextracted into hydrochloric acid. The dried samples were then analyzed by the fluorimetric thiochrome method. Phosphorylated thiamine was treated with Takadiastase to split off the phosphate groups and was thereafter extracted and measured. No column chromatography was needed for the separation of thiamine before the thiochrome assay. A higher accuracy and sensitivity for the thiamine assay was thus achieved.