Utility of Hantzsch reaction for development of highly sensitive spectrofluorimetric method for determination of alfuzosin and terazosin in bulk,dosage forms and human plasma

A highly sensitive, cheap, simple and accurate spectrofluorimetric method has been developed and validated for the determination of alfuzosin hydrochloride and terazosin hydrochloride in their pharmaceutical dosage forms and in human plasma. The developed method is based on the reaction of the primary amine moiety in the studied drugs with acetylacetone and formaldehyde according to the Hantzsch reaction, producing yellow fluorescent products that can be measured spectrofluorimetrically at 480 nm after excitation at 415 nm. Different experimental parameters affecting the development and stability of the reaction products were carefully studied and optimized. The fluorescence–concentration plots of alfuzosin and terazosin were rectilinear over a concentration range of 70–900 ng ml^?1, with quantitation limits 27.1 and 32.2 ng ml^?1 for alfuzosin and terazosin, respectively. The proposed method was validated according to ICH guidelines and successfully applied to the analysis of the investigated drugs in dosage forms, content uniformity test and spiked human plasma with high accuracy.     

Hantzsch condensation reaction as a spectrofluorometric method for determination of alogliptin,an antidiabetic drug,in pure form,tablet form,and human and rat plasma

To analyze alogliptin in its pharmaceutical dosage forms and human plasma, a sensitive, inexpensive, simple, and precise spectrofluorimetric method was developed and tested. This method was also used to investigate the drug’s pharmacokinetic behaviour in the blood of rats. This was based on the Hantzsch reaction, which produces yellowish luminous products that can be detected spectrofluorometrically at 480 and 415 nm for emission and excitation, respectively, when the primary amine group in the examined drug reacts with acetylacetone and formaldehyde. Several experimental parameters that affect the reaction product's development and stability were explored and improved. The curve of fluorescence and concentration for alogliptin was linear in the concentration range 0.05–3.60 μg ml⁻¹. The proposed approach was validated according to International Council for Harmonization criteria. The method was successfully utilized to evaluate the examined drug in dose formulations and spiked human plasma with high accuracy.     

A new spectrofluorimetric method for determination of trace amounts histamine in human urine and serum

A new spectrofluorimetric method was developed for the determination of trace amounts of histamine in human urine and serum samples. In NaAc–HAc buffer solution of pH 4.0, histamine can react with the acetylacetone–formaldehyde system to produce a fluorescent derivative which emits yellow‐green fluorescence at 476 nm, according to the Hantzsch reaction, and the enhanced fluorescence intensity is in proportion to the concentration of histamine. Optimum conditions for the determination of histamine were also investigated. The dynamic range and detection limit for the determination of histamine is 5.96 × 10^–8–1.50 × 10^–5 mol/L and 4.35 × 10^–8mol/L, respectively. This method is practical and can be successfully applied to determination of histamine in human urine and serum samples. A proposal of the reaction pathway is suggested. Copyright © 2009 John Wiley & Sons, Ltd.     

Removal efficiency and mechanisms of formaldehyde by five species of plants in air-plant-water system

The foliar uptake and transport rates of formaldehyde as well as the abilities of leaf extracts to breakdown formaldehyde were investigated to discuss the formaldehyde removal efficiency and mechanism by five species of plants from air. Results showed that formaldehyde could be transported from air via leaves and roots to rhizosphere water. When exposed to 0.56 mg·m^?3 formaldehyde, the formaldehyde removal rate ranged from 18.64 to 38.47 μg·h^?1g^?1 FW (fresh weight). According to the mass balance in the air–plant–water system, the main mechanism of the formaldehyde loss was its breakdown in plant tissues caused by both enzymatic reaction and redox reaction. Higher oxidation potentials of the leaf-extracts of Wedelia chinensis and Desmodium motorium corresponded well to higher abilities to breakdown added formaldehyde than other plants. Based on the different abilities of fresh and boiled leaf-extracts to dissipate formaldehyde, the enzymatic reaction in Chenopodium album L. was the dominant mechanism while the redox reaction in Kochia scoparia (L.) Schrad. and Silene conoidea L. was the main formaldehyde breakdown mechanism when exposed to low-level formaldehyde in air. The redox mechanism suggested that the formaldehyde removal may be increased by an increasing level of reactive oxygen species (ROS) induced by the environmental stress.     

Studies on the bacteriophage MS2. 23. Fixation of the MS2 RNA acid structure by formaldehyde

When MS2 RNA is heated at low p^H in the presence of formaldehyde, a fast-sedimenting conformation is irreversibly formed. This species is homogeneous and stable at neutral p^H. Its formation further requires Mg⁺⁺ ions and low ionic strength. The most compact form sediments at 46S and is obtained after short reaction times at high temperature or after long reaction times at 35°C. Melting curves suggest that the specific acid conformation is not destabilized by the formaldehyde addition reaction. The p^H at which this acid conformation is formed depends on the MgC1₂ concentration. At 10^?2M MgCl₂ the midpoint is p^H 5.3. Removal of more than half of the bound formaldehyde has no effect on the compactness of the molecule, although most of the original secondary structure has not yet re-formed.     

Glyoxal and malonaldehyde formation by ultraviolet irradiation of aqueous formaldehyde

Irradiation of dilute aqueous formaldehyde (5 × 10^?2–10^?3M) in the absence of oxygen by ultaviolet light from high- or low-pressure mercury lamps resulted in the formation of glyoxal and of malonaldehyde. The concentration of malonaldehyde reached a maximum after several hours and then declined. This maximal malonaldehyde concentration was proportional to the initial formaldehyde concentration. At initially 0.05 M formaldehyde (pH 9.4 and 36°C) malonaldehyde reached maximally 3.4 × 10^?5 M. In the range of pH 8.0–11.6, the maximal malonaldehyde concentration was reached at pH 9.4. Quantum yields of glyoxal and malonaldehyde after irradiation of 0.01 M formaldehyde (in 0.01 M NaHCO₃, 27°C, at 254 nm, under argon, for 195 min) were 7 × 10^?3 and 1.5 × 10^?3, respectively. In the presence of acetone (0.01 M), the chemical and quantum yields of glyoxal were enhanced, while those of malonaldehyde decreased. The known reaction of malonaldehyde with urea to form pyrimidines may be a model of a prebiotic synthesis of pyrimidines.     

Formaldehyde as a pre-treatment for dermal collagen heterografts

The preparation, stability both in vitro and in vivo and resistance to bacterial collagenase of trypsin-purified pig dermal collagen cross-linked with a range of concentrations of formaldehyde in phosphate-buffered saline, was studied using ¹⁴C-labelled formaldehyde as a tracer. Washing in phosphate-buffered saline at 37°C produced rapid loss of formaldehyde over 6 weeks before stability was reached. After 19 weeks washing, 12–20% of the initial radioactivity remained, representing 6, 18 and 35 μmol formaldehyde/g of collagen after 21 days reaction with 0.1, 1 and 5% formaldehyde, respectively. Collagen, incorporating stable-bound formaldehyde arising from reaction with formaldehyde in concentrations of 0.5% or over, was totally resistant to bacterial collagenase.The stabilizing effect of formaldehyde cross-linking was also demonstrated by implants of fibrous pig dermal collagen in rats. After 8 weeks a significant constant amount of formaldehyde was retained in all implants. There was no net loss of mass over a 24 week period when pre-treated with 1% formaldehyde but some loss when pre-treated with 0.1% formaldehyde.     

Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca²⁺-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca²⁺-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca²⁺ was replaced by Mg²⁺ or Mn²⁺ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca²⁺-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca²⁺-specific-ATPase luminal localization explains the stable Ca²⁺ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.     

Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride

A procedure is described for radiolabeling proteins in vitro by reductive alkylation. The proteins are treated with [¹⁴C]formaldehyde in the presence of sodium cyanoborohydride, a reducing agent that is stable in aqueous solution at pH 7. The advantage of this procedure is that the reaction can be carried out at neutral pH for extended periods of time and over a wide range of temperatures (0–37°C). Moreover, owing to the flexibility in the reaction conditions afferded by the use of sodium cyanoborohydride, higher incorporation of radiolabel into protein can be attalned.     

Formaldehyde Production by Cells of a Mutant of Candida boidinii S2 Grown in Methanol-limited Chemostat Culture

Formaldehyde production was investigated with cells of a mutant, AOU-1, of a methanol yeast, Candida boidinii S2 grown in methanol-limited chemostat culture. The highest productivity was shown with cells from the culture at a dilution rate of 0.075 hr^-1, when cells had the highest activity of alcohol oxidase and almost minimum activity of formaldehyde dehydrogenase. Under optimal reaction conditions, 950 mm formaldehyde was produced in 10-hr reaction with the cells. By the chemostat culture, not only formaldehyde productivity but also cell productivity was improved in comparison with batch culture. A maximum cell productivity of 0.2 g · liter^-1 · hr^-1 and a cell yield of 47% were obtained.     

Formaldehyde removal in the air by six plant systems with or without rhizosphere microorganisms

Abstract

Uptake and in-plant transport of formaldehyde by six plants with or without soil microorganisms were investigated. The capabilities of fresh and boiled leaf extracts to dissipate added formaldehyde were also measured to evaluate formaldehyde metabolism in plant tissues. Results show that when the initial formaldehyde level in air was 0.56?±?0.04?mg·m^?3, the removal rate in the plant-only systems varied from 1.91 to 31.8?μg·h^?1·g^?1 FW (fresh weight). The removal rate of plants in the plant-only systems were ordered as Helianthus annuus Linn > Lycopersicon esculentum Miller > Oryza sativa > Sansevieria trifasciata Prain > Bryophyllum pinnatum > Mesembryanthemum cordifolium L. f. Most reduction of formaldehyde in the air was due to degradation by active components in the plant tissues, of which 4–64% of these were through to be enzymatic reactions. In the microbe-plant systems, formaldehyde removal rates increased by 0.24–9.53 fold compared to the plant-only systems, with approximately 19.6–90.5% of the formaldehyde reduction resulting from microbial degradation. Microorganisms added to the rhizosphere solution enhanced phytoremediation by increasing the downward transport of formaldehyde and its release by roots. Results suggest a new means to screen for efficient plant species that can be used for phytoremediation of indoor air.     

Antagonistic reactions of arginine and lysine against formaldehyde and their relation to cell proliferation,apoptosis, folate cycle and photosynthesis

¹H, ¹³C NMR, ESMS and MS/MS investigations proved that there is an antagonism in the spontaneous reaction of formaldehyde with L-lysine and L-arginine. L-Arginine can only be hydroxymethylated on the guanidino group in a very fast reaction forming mono-, di-, and trihydroxymethyl arginines (HMA). L-Lysine can be methylated on the -amino group forming mono-, di-, and trimethyl lysine on physiological pH. Hydroxymethyl arginines are relative stable, isolable products, and can also be formed in biological systems, especially in plants. Significant amounts of hydroxymethyl arginines were identified in the aqueous extract of lyophilized kohlrabi, which can be formed in photosynthesis during CO₂ fixation. ¹⁴C-Formaldehyde formed in a short-term (10, 30 sec) ¹⁴CO₂ fixation reaction in Zea mays L. (early maturity variety: Szegedi TC 277) was captured by L-arginine, which occurs in leaves in large amount. Formaldehyde formed during photosynthesis can react not only with the arginine, but with ribulose-1,5-diphosphate present in leaves. In model reactions formaldehyde can react with the ene diole group of ribulose-1,5-diphosphate in the absence of Rubisco enzyme, which is a similar reaction to the addition of formaldehyde to L-ascorbic acid. Hydroxymethyl arginines (HMA) are endogenous formaldehyde carrier molecules transferring the bound formaldehyde to thymidylate synthase enzyme system incorporating it into the folate cycle. HMA can also carry the bound formaldehyde to the cells especially to the tumorous cells (HT29 adenocarcinoma), and cause significant inhibition of cell proliferation and causes apoptosis.     

Oxidation of C1-compounds in Pseudomonas C

Extracts of Pseudomonas C grown on methanol as sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD⁺) and formate dehydrogenase (NAD⁺) were also detected in the extracts.The addition of d-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD⁺ or NADP⁺. In addition, the oxidation of [¹⁴C]formaldehyde to CO₂, by extracts of Pseudomonas C, increased when d-ribulose 5-phosphate was present in the assay mixtures.The amount of radioactivity found in CO₂, was 6.8-times higher when extracts of methanol-grown Pseudomona C were incubated for a short period of time with [1-¹⁴C]glucose 6-phosphate than with [U-¹⁴C]glucose 6-phosphate.These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO₂ both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Why is the molybdenum-substituted tungsten-dependent formaldehyde ferredoxin oxidoreductase not active? A quantum chemical study

Formaldehyde ferredoxin oxidoreductase is a tungsten-dependent enzyme that catalyzes the oxidative degradation of formaldehyde to formic acid. The molybdenum ion can be incorporated into the active site to displace the tungsten ion, but is without activity. Density functional calculations have been employed to understand the incapacitation of the enzyme caused by molybdenum substitution. The calculations show that the enzyme with molybdenum (Mo-FOR) has higher redox potential than that with tungsten, which makes the formation of the Mo^VI=O complex endothermic by 14 kcal/mol. Following our previously suggested mechanism for this enzyme, the formaldehyde substrate oxidation was also investigated for Mo-FOR using the same quantum-mechanics-only model, except for the displacement of tungsten by molybdenum. The calculations demonstrate that formaldehyde oxidation occurs via a sequential two-step mechanism. Similarly to the tungsten-catalyzed reaction, the Mo^VI=O species performs the nucleophilic attack on the formaldehyde carbon, followed by proton transfer in concert with two-electron reduction of the metal center. The first step is rate-limiting, with a total barrier of 28.2 kcal/mol. The higher barrier is mainly due to the large energy penalty for the formation of the Mo^VI=O species.     

Dehalogenation of dichloromethane by cell extracts of hyphomicrobium DM2

A facultatively methylotrophic bacterium was isolated from enrichment cultures containing dichloromethane as the sole carbon source. It was identified as a Hyphomicrobium species. The organism grew exponentially in batch cultures with 10 mM dichloromethane at a specific growth rate of 0.07 h^-1. The release of Cl^- from dichloromethane and the disapperance of substrate paralleled growth. Resting dichloromethane-grown cells, in the presence of potassium sulphite as a trapping agent, converted cichloromethane methane quantitatively to formaldehyde. The conversion of dichloromethane to formaldehyde by cell extracts was stricly dependent on glutathione. Other thiols were inactive. Glutathione was not consumed in the course of the reaction. The specific activity of the enzymic dehalogenation of dichloromethane amounted to 3.8 mkat/kg protein in extracts of dichloromethane-grown cells and to less than 0.1 mkat/kg protein in extracts from cells grown on methanol.     

Highly efficient and selective methoxymethylation of alcohols and phenols catalyzed by high-valent tin(IV) porphyrin

An efficient and selective method for methoxymethylation of alcohols and phenols with formaldehyde dimethyl acetal (FDMA) catalyzed by electron deficient tin(IV)tetraphenylporphyrinato trifluoromethanesulfonate, [Sn^IV(TPP)(OTf)₂], is reported. A variety of primary, secondary and tertiary alcohols as well as phenols were converted to their corresponding methoxymethyl ethers with FDMA in the presence of a high-valent tin(IV) porphyrin. This catalyst can be used for selective methoxymethylation of primary, secondary and tertiary alcohols in the presence of phenols or tertiary alcohols. The present method offers several advantages such as short reaction times, high yields, simple procedure, selectivity and applicability for both alcohols and phenols.     

Carbon‐11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet‐Spengler reaction using carbon‐11 formaldehyde

A procedure for the synthesis of a¹¹C‐labeled oligopeptide containing [1‐¹¹C]1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ([1‐¹¹C]Tpi) from the corresponding Trp?HCl‐containing peptides has been developed involving a Pictet‐Spengler reaction with [¹¹C]formaldehyde. The synthesis of [1‐¹¹C]Tpi from Trp and [¹¹C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon‐11. The Pictet‐Spengler reaction of Trp and [¹¹C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1‐¹¹C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet‐Spengler reaction to an aqueous solution of Trp?HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg‐Gly‐Asp‐D‐Tyr‐Lys] was then selected as a substrate for the labeling reaction with [¹¹C]formaldehyde. The radiolabeling of a Trp?HCl‐containing RGD peptide using the Pictet‐Spengler reaction was successful. Furthermore, the remote‐controlled synthesis of a [1‐¹¹C]Tpi‐containing RGD peptide was attempted by using an automatic production system to generate [¹¹C]CH₃I. The radiochemical yield of the [1‐¹¹C]Tpi‐containing RGD at the end of synthesis (EOS) was 5.9 ± 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 ± 9.4 GBq/µmol at the EOS. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymic method for the quantitative determination of reduced glutathione

A new specific and sensitive assay method for reduced glutathione has been developed. It is based on the reaction HCHO + NAD⁺ + H₂O → HCOOH + NADH + H⁺, catalyzed by formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC.1.2.1.1) in the presence of reduced glutathione. Oxidized glutathione and other thiols do not interfere in this reaction. A purification procedure for formaldehyde dehydrogenase from beef liver is presented.The influence of cysteine and some other thiols, leucine, ascorbate, lactate, pyruvate and four acids generally used for deproteinization is reported. The results obtained by this method from human blood and rat tissues are compared with those obtained by Ellman's method.     

Protein labeling by reductive methylation with sodium cyanoborohydride: Effect of cyanide and metal ions on the reaction

Previous studies (N. Jentoft and D. G. Dearborn, 1979, J. Biol. Chem.254, 4359) have demonstrated that reductive methylation with labeled formaldehyde and NaCNBH₃ provides a simple method for specifically labeling the amino groups of proteins using extremely mild reaction conditions. However, cyanide, which is one of the products of the reaction, reduces labeling effciency by reacting with formaldehyde to from the formaldehyde cyanohydrin addition product. Certain transition metal ions are able to prevent this secondary reaction by forming stable coordination complexes with cyanide. Inclusion of millimolar quantities of Ni(II) in reaction mixtures leads to a 20–30% increase in protein labeling so that maximal derivatization of amino groups can be realized with only a 3- to 4-fold ratio of formaldehyde to amine rather than the 5- to 10-fold excess necessary in the absence of metal ions.     

Large-Scale and Small-Scale Methods for the Preparation of 5,10-Methylenetetrahydrofolate

Abstract

Two procedures have been developed for the synthesis and isolation of 5,10-methylenetetrahydrofolate, the cofactor for the reaction catalyzed by thymidylate synthesize, one of which can be used for large-scale preparations of the cofactor and the other for small-scale syntheses especially suitable for obtaining the radio labeled cofactor. The large-scale procedure involves treatment of folic acid with dithionite to give dihydrofolate, which is then converted to tetrahydrofolate by dihydrofolate reductase (L. casei). The small-scale method involves a direct enzymatic reduction of folic acid to tetrahydrofolate by dihydrofolate reductase, and has been used to prepare the double-labeled 5,10-[¹⁴C]methylene[3′,5′,7,9-³H]tetrahydrofolate. In both procedures, after the reduction steps have been performed, the tetrahydrofolate is treated in situ with formaldehyde prior to purification by DEAE-cellulose chromatography, thus allowing the isolation of 5,10-methylenetetrahydrofolate as a dry powder after lyophilization. This product is active in the enzyme reaction without the further addition of excess formaldehyde as in previous procedures. The cofactor prepared in this manner has much improved stability toward oxidation compared to free tetrahydrofolate.