Isolation and partial characterization of a 51Cr complex from Brewers' yeast

A butanol: H₂O extract of Brewers' yeast grown in a medium which contained ⁵¹Cr was analyzed by gel-filtration chromatography. A single radioactive peak was eluted at an elution volume which suggested a molecular weight of approximately 400–600 daltons. Subsequent examination of pooled radioactive fractions obtained from gel-filtration chromatography demonstrated that the ⁵¹Cr complex was eluted in a single peak from both cation- and anion-exchange resins. The elution characteristics of the ⁵¹Cr complex indicated that the compound is a single anionic species. The ⁵¹Cr complex was purified by a combination of gel-filtration chromatography and ion-exchange chromatography and subsequently analyzed by thin-layer chromatography. The results indicated that the ⁵¹Cr complex from Brewers' yeast is a peptide that contains at least six amino acids. When a partially purified preparation of the ⁵¹Cr complex from yeast was administered orally to rats, the absorption and retention of ⁵¹Cr was significantly greater than that in rats given ⁵¹Cr in the form of CrCl₃·6H₂O. These experiments demonstrate that chromium is associated with a single metal-binding peptide in Brewers' yeast and indicate that the chromium in this complex is absorbed and retained more efficiently than chromium salts.     

Chromium uptake bySaccharomyces cerevisiae and isolation of glucose tolerance factor from yeast biomass

Fermentations with yeastSaccharomyces cerevisiae in semiaerobic and in static conditions with the addition of chromic chloride into the used molasses medium were analysed. It was proved that the addition of optimal amounts of CrCl₃ into the basal medium enhanced the kinetics of alcohol fermentations. The addition of 200 mg/l CrCl₃ into the medium stimulated both the yeast growth and the ethanol production in all experimental conditions. On the other hand, the results showed that Cr³⁺ ions were incorporated into yeast cells during fermentation. Under these conditions the accumulation of Cr³⁺ ions was performed by yeast cells during the exponential growth phase, and with enriched amounts of 30–45 (μg/g_d.m. of cells. Yeast biomass enriched with chromium ions was extracted with 01 mol/l NH₄OH assuming that the extracts had the glucose tolerance factor (GTF). Then the extracts were passed through a gel-filtration column in order to isolate and purify the GTF. The presence of GTF in the purified fractions was determined by measuring the absorbance at 260 nm. It is evident from the obtained results that the added purified fractions enhanced the rates of CO₂ production as well as the glucose utilization during alcoholic fermentation. As expected, the enhancement of both rates depended on the amounts of extracts added to the fermentation substrate. Thus, it is evident that purified extracts contained the GTF compound, and that Cr³⁺ ions were bonded to the protein molecule.     

An investigation by ion-exchange chromatography of a chromium,amino acid and nicotinic acid mixture

The mixture of chromium, nicotinic acid and the amino acids glycine, glutamic acid and cysteine which stimulates the rate of CO₂ production in a yeast bioassay system was subjected to the separation scheme based on ion-exchange chromatography which has been used to separate the chromium- containing fractions in brewer's yeast, [S.J. Haylock, P.D. Buckley and L.F. Blackwell, J. Inorg. Biochem., 18, 195 (1983)]. Four chromium-containing fractions (C2 to C5) were obtained by salt gradients and two further fractions (G1 and G2) were obtained using a pH gradient. All were amino acid-containing complexes of chromium and all except C5 also contained nicotinic acid. However, none of the isolated chromium fractions showed any activity in a yeast bioassay. On the basis of previous work, the activity of the original mixture was attributed to the presence of an oxygen-coordinated trans chromium(III)-dinicotinate complex. Biologically- inactive chromium complexes such as Cr(glu)₂(H₂O)⁺₂ and Cr(gly)₂(H₂O)⁺₂ after elution by ammonium hydroxide from Dowex 50W-X12 cation- exchange columns, stimulated the rate of CO₂ production in the yeast bioassay. Elution with other bases, such as lithium hydroxide, potassium hydroxide and sodium hydroxide led to inactive fractions in all cases. A warning is therefore given that the use of ammonium hydroxide-elution of ion-exchange columns to isolate glucose tolerance factor fractions from biological samples (such as brewer's yeast) can lead to active fractions which do not relate to the native material.     

Hypoglycemic activity and acute oral toxicity of chromium methionine complexes in mice

The hypoglycemic activity of chromium methionine (CrMet) in alloxan-induced diabetic (AID) mice was investigated and compared with those of chromium trichloride hexahydrate (CrCl₃·6H₂O) and chromium nicotinate (CrNic) through a 15-day feeding experiment. The acute oral toxicity of CrMet was also investigated in ICR (Institute for Cancer Research) mice by a single oral gavage. The anti-diabetic activity of CrMet was explored in detail from the aspects of body weight (BW), blood glucose, triglyceride, total cholesterol, liver glycogen levels, aspartate transaminase (AST) and alanine transaminase (ALT) levels. The obtained results showed that CrMet had beneficial effects on glucose and lipid metabolism, and might possess hepatoprotective efficacy for diabetes. Daily treatment with 500 and 1000 μg Cr/kg BW of CrMet in AID mice for 15 days indicated that this low-molecular-weight organic chromium complex had better bioavailability and more beneficial effects on diabetics than CrCl₃·6H₂O. CrMet also had advantage over CrNic in the control of AST and ALT activities. Acute toxicity studies revealed that CrMet had low toxicity potential and relatively high safety margins in mice with the LD₅₀ value higher than 10.0 g/kg BW. These findings suggest that CrMet might be of potential value in the therapy and protection of diabetes.     

Separation of biologically active chromium-containing complexes from yeast extracts and other sources of glucose tolerance factor (GTF) activity

A procedure has been developed, based on ion-exchange chromatography, that readily allows the separation of eleven apparently homogeneous chromium-containing fractions from a brewer's yeast extract. Four of the fractions are amphoteric and show no glucose tolerance factor (GTF) activity, three are classified as negative (two of which are biologically inactive, while the third one shows a slight degree of GTF activity), whereas the four cationic chromium-containing fractions all show varying degrees of GTF activity. Application of the separation procedure to other biological sources of GTF activity resulted in a spectrum of cationic fractions, over the pH range 1.75 to 12, which suggests that GTF cannot be a single species. The cationic chromium-containing fraction from pork kidney powder and fraction P-3 from yeast appear to contain the most GTF-active material and P-3 shows saturation kinetics as expected for a biologically significant substance.     

Anti-hyperglycemic Activity of Chromium(III) Malate Complex in Alloxan-Induced Diabetic Rats

The synthesis, characterization, anti-hyperglycemic activity, oxidative DNA damage capacity, and acute toxicity of chromium(III) malate complex [Cr₂(LMA)₃] were described. [Cr₂(LMA)₃] was synthesized in a single-step reaction by chelating chromium(III) with L-malic acid in aqueous solution. Based on elemental analysis, thermodynamic analysis, and spectroscopy studies, the molecular formula of [Cr₂(LMA)₃] was inferred as Cr₂(C₄H₄O₅)₃·5H₂O. Daily treatment with 2.85–17.10 mg/kg body mass of [Cr₂(LMA)₃] in alloxan-induced diabetic rats for 2 weeks indicated that low-molecular-weight organic chromium complex [Cr₂(LMA)₃] had better bioavailability and more beneficial influences on the improvement of controlling blood glucose, serum lipid, and liver glycogen levels compared with CrCl₃·6H₂O. [Cr₂(LMA)₃] did not cause oxidative DNA damage under physiologically relevant conditions. Acute toxicity studies revealed no-measurable toxicity of the [Cr₂(LMA)₃]. Collectively, these results suggest that [Cr₂(LMA)₃] may represent a novel, proper chromium supplement with potential therapeutic value to control blood glucose and serum lipid in diabetes.     

The influence of chromium chloride consumption on lipid peroxidation and activity of the antioxidant defense in rat tissues

The effect of food supplementation with chromium (CrCl₃ · 6H₂O) on intensity of peroxide processes and activity of antioxidant enzymes has been investigated in some rat tissues. Food supplementation with 200 μg/kg CrCl₃ · 6H₂O for 30 days resulted in the increase of tissue chromium. The tissue chromium content of chromium-treated rats decreased in the following order: spleen, heart, kidney, lung, brain, liver, skeletal muscles. All organs and tissues (except skeletal muscles) of chromium-treated rats were characterized by decreased content of lipid peroxidation (LPO) products: hydroperoxides and thiobarbituric acid reactive substances (TBARS). The maximal reduction in LPO products was observed in spleen, kidney, liver, and lung. Treatment with chromium also caused an increase in the activity of glutathione peroxidase, glutathione reductase, and calatase in all tissues and organs studied. In the brain and kidney an increase in the content of reduced glutathione was observed. Superoxide dismutase activity was higher in myocardium and skeletal muscles, basically equal in lung and liver, while in other organs (brain, kidney, spleen) of experimental animals it was lower than in control animals. Results of this study suggest that chromium exhibits tissue/organ-specific regulatory effects on enzymes of the antioxidant defense     

Effects of chromium compounds on plasma membrane Mg2+-ATPase activity of BHK cells.

An ATPase activity stimulated by divalent ions (Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺) has been observed in intact hamster fibroblasts cultured in vitro (BHK line). Such activity has been determined by the incubation (30 min at 37°C) of washed cell suspensions (about 1 mg of proteins) in a medium containing 100 mM NaCl, 20 mM KCl, 15 mM Tris—HCl (pH 7.4), 10 mM NaHCO₃, 5 mM glucose and equimolar concentrations of ATP and divalent cation. Mg²⁺-ATPase activity is insensitive to ouabain and lacks specificity towards nucleoside triphosphate substrates. AMP and ADP are not hydrolyzed under these conditions. Apparent K_m of 0.76 mM and V_max of 1.46 μmol Pi · mg proteins^?1 · h^?1 have been calculated for Mg-ATP complex. This ATPase is an ectoenzyme, therefore its activity could be used as a suitable index of the action of chemicals like chromium compounds known for their cytotoxic effects on membrane functions.Salts of trivalent (CrCl₃) and hexavalent (K₂Cr₂O₇) chromium at concentrations ranging from 1 mM to 5 mM inhibit Mg²⁺-ATPase. The inhibition by K₂Cr₂O₇ is observed after pretreatment of the cells with this compound followed by its absence from the assay medium “per se” for Mg²⁺-ATPase, and it is referred to the alterations of membrane bound enzyme structures by the oxidizing hexavalent chromium. The inhibition by CrCl₃ is mainly evident when this compound is present in the incubation medium, and is referred to the interaction of trivalent chromium with Mg²⁺-ATP as it is partially reversed by increasing Mg²⁺-ATP concentration.     

Chromium Improves Protein Deposition Through Regulating the mRNA Levels of IGF-1, IGF-1R,and Ub in Rat Skeletal Muscle Cells

The aim of this study was to evaluate the impact of three different chromium forms—chromic chloride (CrCl₃), chromium picolinate (CrPic), and a newly synthesized complex of chromium chelated with small peptides (CrSP)—on protein metabolism in vitro. In cultured skeletal muscle cells, CrSP was able to increase the basal and insulin-stimulated levels of protein deposition in skeletal muscles cells. CrCl₃ and CrPic augmented insulin-stimulated protein synthesis. At the molecular level, insulin significantly increased the mRNA levels of insulin-like growth factor 1 and insulin-like growth factor 1 receptor. These impacts could be enhanced by the addition of chromium, especially CrSP. The mRNA levels of ubiquitin were significantly reduced when cells were cultured with chromium or/and insulin. Assuming that the mRNA level increase or decrease results in increased or decreased levels of these proteins, chromium would improve protein anabolism and reduce protein catabolism and then prove protein deposition in rat skeletal muscle cells.     

The isolation of glucose tolerance factors from brewer's yeast and their relation to chromium

A new dietary factor, the glucose tolerance factor (GTF), was reported in 1957 that improved impaired glucose tolerance in rats. Most studies on GTF have used brewer's yeast as the starting material, and it has been postulated that the active material is a low-mol wt organic complex containing Cr³⁺. It seemed thus important to isolate an active GTF from chromium-rich yeast (228 ppm Cr) obtained by incubation with chromium and to compare each fraction with corresponding ones from untreated yeast (0.48 ppm Cr). We developed an isolation and purification procedure by fractionation of yeast extract on an anion and cation exchange resin, and tested the GTF activity (glucose oxidation) on rat adipocytes. PIXE (proton-induced X-ray emission) was used to measure the chromium content of the individual fraction. Individual fractions with GTF activity did not differ between Cr-rich and Cr-deficient yeast, and there was no relationship between Cr content and GTF activity. This does not support the hypothesis that chromium is an obligatory constituent of the GTF, assuming that GTF is a unique substance.     

Effects of dietary chromium supplementation on performance, carcass traits, serum metabolites, and tissue chromium levels of Japanese quails

This study was conducted to investigate the effects of various levels of dietary chromium supplementation on performance, carcass traits, blood chemistry, and tissue distribution of chromium (Cr³⁺) in quails. Two hundred forty 1-d-old Japanese quails were divided into five groups with four replicates and were fed a basal diet or the basal diet supplemented with 20, 40, 80, or 100 mg/kg Cr (CrCl₃·6H₂O) until 38 d of age. Chromium supplementation decreased carcass fat percentage, serum low-density lipoprotein (LDL), and glucose and increased serum magnesium (Mg) and Cr content of kidney, liver, and muscle. In conclusion, 20, 40, 80, or 100 mg/kg Cr supplementation to quail diet had no effect on performance, chemical composition of carcass except fat percentage, serum protein, calcium (Ca), and inorganic phosphorus (Pi) levels, but reduced serum glucose, LDL and fat percentage of carcass. Chromium is accumulated mainly in the kidneys and liver.     

A new medium for spore production of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> using response surface methodology

Optimization of the medium components which enhance sporulation of the two mating types of the fungus Blakeslea trispora ATCC 14271 and ATCC 14272 (a heterothallic Zygomycota producing carotene) was achieved with the aid of response surface methodology (RSM). Glucose, corn steep liquor, yeast extract, and ammonium sulfate were investigated as carbon and nitrogen sources in a basal medium. RSM was adopted to optimize the medium in order to obtain a good growth of the fungus as a prerequisite for enhanced sporulation. In the second step, the basal medium was supplemented with different trace elements which significantly affect sporulation (i.e. CuSO₄·5H₂O, FeCl₃·6H₂O, Co(NO₃)₂·6H₂O, and MnCl₂·4H₂O). Central composite design proved to be valuable in optimizing a chemically defined solid medium for spore production of B. trispora. The composition of the new solid medium to enhance spore production by B. trispora (ATCC 14271) is as follows (per liter): 7.5 g glucose, 3.2 g corn steep liquor, 1.7 g yeast extract, 4.1 g ammonium sulfate, 6 mg CuSO₄·5H₂O, 276 mg FeCl₃·6H₂O, 2 mg Co(NO₃)₂·6H₂O, and 20 g agar (pH 6.0). Practical validation of this optimum medium gave spore number of 1.2 × 10⁸ spores/dish which is 77% higher than that produced in Potato Dextrose Agar (PDA). In the case of B. trispora (ATCC 14272) the new solid substrate for enhanced sporulation consists of (per l) 6.4 g glucose, 3.3 g corn steep liquor, 1.4 g yeast extract, 4.3 g ammonium sulfate, 264 mg CuSO₄·5H₂O, 485 mg FeCl₃·6H₂O, 223 mg MnCl₂^.4H₂O, and 20 g agar (pH 6.0). Spore numbers of 2 × 10⁷ spores/dish were obtained on the new medium by B. trispora (ATCC 14272), which is 95% higher than that produced on PDA. The results corroborated the validity and the effectiveness of the models. The new media considerably improved sporulation of both strains of B. trispora compared to the production of spores on PDA, which is the medium usually used for sporulation of the fungus.     

Catalytic conversion of glucose to 5-hydroxymethyl furfural using inexpensive co-catalysts and solvents

Efficient conversion of glucose to 5-hydroxymethyl furfural (5-HMF), a platform chemical for fuels and materials, was achieved using CrCl₂ or CrCl₃ as the catalysts with inexpensive co-catalysts and solvents including halide salts in dimethyl sulfoxide (DMSO) and several ionic liquids. 5-HMF (54.8%) yield was achieved with the CrCl₂/tetraethyl ammonium chloride system at mild reaction conditions (120 °C and 1 h). The 5-HMF formation reaction was found to be faster in ionic liquids than in the DMSO system. Effects of water in the reaction system, chromium valence and reaction temperature on the conversion of glucose into 5-HMF were discussed in this work.     

Chromium(III) complexes and their relationship to the glucose tolerance factor. Part II. Structure and biological activity of amino acid complexes

A number of octahedral chromium complexes with amino acids are ligands have been prepared and their structures assigned on the basis of their chromatographic and spectral properties. These include complexes with the general structure Cr(AA)₂(H₂O)₂ where the amino acids glycine, glutamic acid and glutamine act as bidentate ligands. The analogous compound with cysteine as ligand is stable at low pH, but at high pH a terdentate cysteine complex, Cr(cysteine)₂^?, is formed. These complexes, as well as a solution of monodentate glycine aquo complexes, and Cr-nicotinic acid-glycine and Cr-nicotinic acid-cysteine complexes of undetermined structure, have been assayed for glucose tolerance factor activity using a yeast assay. Only Cr(glutamine)₂- (H₂O)₂⁺, Cr-nicotinic acid-glycine and the mixture of complexes Cr(glycine)_n(H₂O)_6-n⁺³ showed significant activity. It is proposed that a trans arrangement of the non-coordinated nitrogen atoms in the ligands of these complexes can mimic the structural features of the glucose tolerance factor which are essential for biological activity.     

A study of some solvolysis reactions of bis(trimethylamine)chromium(III) chloride

Two competing reactions are present when benzene solutions of the five co-ordinate complex CrCl₃·2NMe₃ are treated with donor molecules viz., a) ligand substitution via solvolysis of metal-nitrogen bonds and b) the independent decomposition of the bis-trimethylamine adduct into tri-μ-chloro-trichlorotris(trimethylamine) dichromium(III), Cr₂Cl₆(NMe₃)₃, and trimethylamine. For all but very feeble donors reaction a) predominates and in the ensuing adduct formation the chromium(III) ions assume hexacoordination, e.g. pyridine and tetrahydrofuran react immediately to give the corresponding CrCl₃·3L complexes. Reaction b) shows second order kinetics with a rate constant k = 0.160 1 mole⁻¹ sec⁻¹ The spectral and magnetic properties of the binuclear compound Cr₂Cl₆(NMe₃)₃ have been interpreted in terms of adjacent six co-ordinate metal atoms and a proposed structure is based on two fused octahedra sharing a common trigonal face. The product obtained on treatment of CrCl₃·2NMe₃ with AgClO₄ involves only bidentate (C_2v) perchlorate ions as co-ordinated ligand and is formulated as Cr(ClO₄)₃.     

Effects of C/N ratio and trace elements on mycelial growth and exo-polysaccharide production of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>

Although its demand increased greatly due to the volatile strong flavor and bioactive molecules, little information has been about the cultural characteristics of Tricholoma matsutake. In this study, we investigated the optimal medium composition of liquid culture with the goal of shortening the culture period, and to maximize polysaccharide production and mycelial growth. From these experiments we found that the optimal medium contained 40 g/L, glucose; 30 g/L, yeast extract; 1.5 g/L, KH₂PO₄; and 1 g/L MgSO₄·7H₂O. In flask culture, the maximum mycelial growth and polysaccharide production were 22.45 and 5.3 g/L, which were about 9 and 3 g/L higher than that at the basal medium, respectively.     

Determination of an economical medium for growth of Lactobacillus fermentum using response surface methodology

Aim: Lactobacillus fermentum is a widely utilized probiotic compound fed as an alternative to antibiotics for growth promotion in a wide variety of livestock species. The objective of this research is to develop an economical and practical fermentation medium for the growth of Lact. fermentum using response surface methodology. Methods and Results: A two‐level Plackett–Burman design was used to determine which factors in the fermentation medium influence the growth of Lact. fermentum. Under our experimental conditions, peptone, urea and yeast extract were found to be major factors. Then, the steepest ascent method and the central composite design were applied to optimize the culture of Lact. fermentum. The following composition of the fermentation medium was estimated to be the most economical formula (per litre): 30 g corn syrup, 15 g glucose, 14·4 g peptone, 7 g (NH₄)₂SO₄, 0·5 g urea, 3 g sodium acetate, 4 g sodium citrate, 0·1 g MnSO₄·4H₂O, 0·5 g MgSO₄·7H₂O, 7·3 g yeast extract, 0·5 g K₂HPO₄. Conclusion: Based on 10 side‐by‐side comparisons, we found that the yield of Lact. fermentum using our fermentation medium was 64% greater than those using modified de Man, Rogosa and Sharp broth (MRS) medium (1·8 × 10⁹ CFU ml^?1vs 1·1 × 10⁹ CFU ml^?1, respectively), while the cost was 89% lower than MRS. This research indicates that it is possible to increase bacterial yield by using inexpensive materials. Significance and Impact of the Study: It is more likely that the use of Lact. fermentum as a probiotic will increase. The low cost medium developed in this research can be used for large‐scale, commercial application where economics are quite likely to be important.     

Absorption, excretion and retention of 51Cr from labelled Cr-(III)-picolinate in rats

The bioavailability of chromium from Cr-picolinate (CrPic₃) and Cr-chloride (CrCl₃) was studied in rats using ⁵¹Cr-labelled compounds and whole-body-counting. The intestinal absorption of Cr was twice as high from CrPic₃ (1.16% vs 0.55%) than from CrCl₃, however most of the absorbed ⁵¹Cr from CrPic₃ was excreted into the urine within 24 h. After i.v. or i.p. injection, the whole-body retention curves fitted well to a multiexponential function, demonstrating that plasma chromium is in equilibrium with three pools. For CrPic₃, a large pool exists with a very rapid exchange (T _1/2 = <0.5 days), suggesting that CrPic₃ is absorbed as intact molecule, from which the main part is directly excreted by the kidney before degradation of the chromium complex in the liver can occur. CrCl₃ is less well absorbed but the rapid exchange pool is much smaller, resulting in even higher Cr concentrations in tissue such as muscle and fat. However, 1–3 days after application, the relative distribution of ⁵¹Cr from both compounds was similar in all tissues studied, indicating that both compounds contribute to the same storage pool. In summary, the bioavailability of CrPic₃ in rats is not superior compared to CrCl₃.     

Preparation and characterization of some Cr(III) chloro-complexes with nicotinic acid esters

The preparation and characterisation of the trichlorotris(alkylnicotinate)chromium(III) complexes of general formula CrCl₃(py·3COOR)₃·nH₂O, where R = Me, Et, Pr and Bu are reported, n being 3.5, 1.0, 0 and 0 respectively. It is concluded that the ligation of the three chloride ions and that of the three nitrogen atoms is consistent with a C_2u arrangement in each case.     

Chromium(III) interactions with nucleotides. II

New derivatives were obtained from Cr(urea)₆Cl₃· 3H₂O in an ethyl acetate medium of chromium(III) with uracil, uridine, 5′UMP, 5′CMP, 5′GMP and 5′IMP. The new derivatives were characterized by elemental analysis, electronic and infrared spectroscopy and thermal analysis. These derivatives proved to be outer sphere complexes, in which the nucleotide, the nucleoside or the base interacts with the starting complex through intramolecular hydrogen bonding.Cr(XMP)(OH)·3H₂O (XMP: 5′UMP, 5′CMP, 5′GMP and 5′IMP) complexes were obtained by hydrolysis of the above derivatives of the nucleotides. In these reactions there is a total substitution of the urea molecules. The derivatives obtained by hydrolysis were characterized in solid state by electronic and infrared spectroscopy. These results provide more insight into the biological role of chromium.