Measurement of inorganic orthophosphate in biological materials: Extraction properties of butyl acetate

The properties of several organic solvents were investigated to determine their suitability for use in phosphomolybdic acid (PMA) extraction procedures for the measurement of inorganic orthophosphate (P_i). Butyl acetate exhibited the most satisfactory properties for measurements at 310 nm, which is the absorption peak for unreduced PMA. Butyl acetate exhibits essentially no absorption at 310 nm, is highly selective for PMA over molybdate and silicomolybdate, does not extract molybdate in the presence of trichloroacetic acid (TCA), exhibits negligible volume changes when equilibrated with equal volumes of aqueous phase, and is among the least toxic of organic solvents. Optimal conditions of acidity, molybdate concentration, and reaction and extraction times were determined for the formation of PMA and extraction into butyl acetate. A procedure for measurement of P_i in biological material employing 8% TCA for precipitation of organic material at 0°C, reaction of the supernatant with acid molybdate, extraction of the PMA with butyl acetate and reading of unreduced PMA at 310 nm is described. The procedure is simple, rapid, accurate, selective, and has high sensitivity. Because of the short (20 sec) sample exposure to acid molybdate, it is suggested that the procedure may also be useful in the measurement of P_i in the presence of adenosine triphosphate such as for the assay of adenosinetriphosphatase activity.     

Rapid determination of tamsulosin in human plasma by high-performance liquid chromatography using extraction with butyl acetate

A high-performance liquid chromatographic method with fluorescence detection for the determination of tamsulosin in human plasma is reported. The sample preparation involved liquid-liquid extraction of tamsulosin from alkalised plasma with butyl acetate and back-extraction of the drug to the phosphate buffer (pH 2). Butyl acetate is preferable to more commonly used ethyl acetate because of its much lower solubility in water. Liquid chromatography was performed on an octadecylsilica column (55 mm x 4 mm, 3 microm particles), the mobile phase consisted of acetonitrile-30 mM dihydrogenpotassium phosphate (25:75 v/v). The run time was 3.5 min. The fluorimetric detector was operated at 228/326 nm (excitation/emission wavelength). An analogue of tamsulosin, (R)-5-[2-[(3-(2-ethoxyphenoxy)propyl)amino]-2-methylethyl]-2-methoxybenzensulfonamide was used as the internal standard. The limit of quantitation was 0.4 ng/ml using 1 ml of plasma. Within-day and between-day precision expressed by relative standard deviation was less than 10% and inaccuracy did not exceed 5%. The assay was applied to the analysis of samples from several pharmacokinetic studies.     

Determination of tripdiolide in root extracts of Tripterygium wilfordii by solid-phase extraction and reversed-phase high-performance liquid chromatography

Extracts of Tripterygium wilfordii Hook F. have been widely used in China to treat a variety of autoimmune and inflammatory diseases. The diterpenoids triptolide and tripdiolide are two major active components in the T. wilfordii ethyl acetate extract. An efficient solid-phase extraction and high-performance liquid chromatography (SPE-HPLC) method to measure triptolide content in the extract has been previously reported. However, a suitable means of tripdiolide quantification is not available because of interfering compounds in the extract that co-elute with tripdiolide. Therefore, this paper describes a method wherein tripdiolide content can be measured from a small amount of the extract. The extract solution (600 microL) was applied into an aminopropyl SPE tube. Triptolide was eluted with dichloromethane:methanol (1 mL, 49:1 v/v), followed by tripdiolide elution with dichloromethane:methanol (3 mL, 17:3 v/v). The tripdiolide eluate was analysed by HPLC using an isocratic solvent system and was quantified by measuring the peak area at 219 nm. The contents of triptolide and tripdiolide in the extract were determined to be 807.32 +/- 51.94 and 366.13 +/- 17.21 microg/g of extract, respectively. Since tripdiolide is biologically active and makes up a considerable portion of the extract, for extract quality control and standardisation purposes, it should be measured along with triptolide using the proposed SPE-HPLC method.     

Olfaction and neurogenesis in the olfactory epithelium

Anosmia was experimentally produced in strain C57BL/6 laboratory mice by treatment with 1% zinc sulfate solution. Structural and functional changes taking place in the olfactory epithelium were investigated during this process and during reinstatement of olfaction. Isoamyl acetate, butyl acetate, and substances present in murine urine were used as olfactory stimuli. Response to these odorants was found to recover from zinc sulfate action at different rates. The highest (both relative and absolute) daily rise in amplitude response was that induced by isoamyl acetate and butyl acetate and lowest in the case of odors of biological origin. Response to olfactory stimuli recovered most rapidly in the areas of the epithelium where maximum response to the same stimuli had been seen in intact animals."Biopharmautomatica" Combined Research and Production Unit, Gor'kii. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 500–506, July–August, 1990.     

A modified direct phosphate assay for studying ATPases

A simple, rapid assay for purified ATPases is presented, based upon the formation of phosphomolybdate and its extraction into butyl acetate. The inclusion of imidazole makes the assay more sensitive and reproducible apparently because of the formation of an imidazole-phosphomolybdate complex. Protein (100 micrograms), Hepes buffer [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (0.1 M) and nucleotides (1 mM) were all shown to cause interference. The interference by nucleotides could be counteracted by using more molybdate. Butyl acetate was shown to extract virtually all of the phosphomolybdate almost instantaneously upon vortex mixing.     

Destabilization of emulsions during extraction of antibiotics from natural solutions]

Various means for changing the aggregative stability of emulsions in the system of benzylpenicillin fermentation broth filtrate and butyl acetate were tested. It was shown that the use of desemulgators was more efficient than the use of some means for removing the desemulgating admixtures. Moreover, addition of surface active substances after the preliminary thermocoagulation resulted in maximum decreasing of the emulsion aggregative stability.     

Purification of protease-extracted glycosaminoglycans by short-distance paper chromatography with a zinc acetate solvent

A method for the purification of tissue-extracted glycosaminoglycans, using short-distance (5-cm path) paper chromatography with 0.25 m zinc acetate solvent, pH 5.0, is presented. The method removed 96 to 97% of the impurities present in the original tissue extract, based on absorbance at 260 nm and protein and phosphate contents. Recovery of control samples of dermatan sulfate averaged 88%. In place of zinc, manganous and mercuric acetates were used to test the method; however, zinc was found to be preferable.     

Urokinase-type plasminogen activator mediates basic fibroblast growth factor-induced bovine endothelial cell migration independent of its proteolytic activity.

The dependence of urokinase-type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA-dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30-50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28-50%). Anti-bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which is enzymatically inactive, stimulated endothelial cell movement in the presence of anti-bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA-mediated proteolysis.     

千金子不同极性部位对酪氨酸酶活性的影响

测定千金子醇提物不同极性部位对酪氨酸酶活性的影响。以L-酪氨酸为底物,采用比色法测定千金子不同极性部位对酪氨酸酶的抑制率。得到了千金子醇提物的乙酸乙酯相、正丁醇相和水相的半数抑制率(IC50值)分别为0.150 mg/mL,0.813 mg/mL,7.570 mg/mL,并对乙酸乙酯相进一步分离得到七叶内酯,其IC50值0.103 mg/mL。结果表明千金子中起到酪氨酸酶抑制性的物质为七叶内酯,主要分布在乙酸乙酯相中。为千金子中酪氨酸酶抑制性物质的筛选提供科学依据。     

Contraction of fibrillar type I collagen by endothelial cells: A study in vitro

The formation of microvascular sprouts during angiogenesis requires that endothelial cells move through an extracellular matrix. Endothelial cells that migrate in vitro generate forces of traction that compress (i.e., contract) and reorganize vicinial extracellular matrix, a process that might be important for angiogenic invasion and morphogenesis in vivo. To study potential relationships between traction and angiogenesis, we have measured the contraction of fibrillar type I collagen gels by endothelial cells in vitro. We found that the capacity of bovine aortic endothelial (BAE) cells to remodel type I collagen was similar to that of human dermal fibroblasts—a cell type that generates high levels of traction. Contraction of collagen by BAE cells was stimulated by fetal bovine serum, human plasma-derived serum, bovine serum albumin, and the angiogenic factors phorbol myristate acetate and basic fibroblast growth factor (bFGF). In contrast, fibronectin and immunoglobulin from bovine serum, several nonserum proteins, and polyvinyl pyrrolidone (a nonproteinaceous substitute for albumin in artificial plasma) were not stimulatory. Contraction of collagen by BAE cells was diminished by an inhibitor of metalloproteinases (1, 10-phenanthroline) at concentrations that were not obviously cytotoxic. Zymography of proteins secreted by BAE cells that had contracted collagen gels revealed matrix metalloproteinase 2. Subconfluent BAE cells that were migratory and proliferating were more effective contractors of collagen than were quiescent, confluent cells of the same strain. Moreover, bovine capillary endothelial cells contracted collagen gels to a greater degree than was seen with BAE cells. Collectively, our observations indicate that traction-driven reorganization of fibrillar type I collagen by endothelial cells is sensitive to different mediators, some of which, e.g., bFGF, are known regulators of angiogenesis in vivo. © 1996 Wiley-Liss, Inc.     

Improved determination of bovine glutaminyl cyclase activity using precolumn derivatization and reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive, rapid and reproducible assay for the determination of glutaminyl cyclase activity is reported. This method is based on the monitoring of the absorption of l-pyroglutamic acid beta-naphthylamide at 235 nm, enzymatically formed from the substrate l-glutaminyl-beta-naphthylamide, after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.08 nmol/ml and, the time consumed for analysis is <6.5 min per sample for separation and quantification. The optimum pH for glutaminyl cyclase activity was 8.0-8.5. The K(m) and V(max) values were 100.2+/-2.9 microM and 332 +/-21.7 pmol/(h microg protein), respectively, with the use of enzyme extract obtained from bovine pituitary. Glutaminyl cyclase activity was strongly inhibited by zinc(II) ion and 1,10-phenanthroline. By using this assay, the stimulatory effect of bacterial lipopolysaccharide on this enzyme activity was observed in macrophage cell line RAW 264.7. Our newly developed assay would be useful for clarification of the physiological role of this enzyme.     

Determination of epimedin C in rat plasma by reversed-phase high-performance chromatography after oral administration of Herba Epimedii extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile -0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05-4.0 microg/mL. The lower limit of quantification was 0.05 microg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.     

Ferric acetate-uranium acetate reagent in cholesterol estimations in plasma and high density lipoprotein fractions: comparison with existing methods

The use of ferric acetate-uranium acetate colour reaction for the estimation of cholesterol in the supernatants of plasma samples after precipitation of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol by heparin-MnCl2 was assessed and compared with the conventional method using the FeCl3 colour reaction and also with the method using o-phthalaldehyde as the colouring reagent. All three methods gave comparable values when total cholesterol in plasma samples was determined and also when high density lipoprotein (HDL) fractions were separated by ultracentrifugation and the cholesterol contents determined. But when heparin-MnCl2 precipitation was used for HDL separation, and the cholesterol content determined, the FeCl3 method gave significantly lower values. This could be due to interference of the cholesterol colour reaction with FeCl3, due to Mn2+ ions present in the supernatant. Addition of Mn2+ to cholesterol standards and subsequent colour development with ferric acetate-uranium acetate and FeCl3 reagents showed that Mn2+ decreased the absorbancy of the coloured complex at 560 nm only when FeCl3 was used. Percentage recovery of added cholesterol was also lower when the heparin-MnCl2 supernatant was treated with FeCl3 reagent for colour development. Use of ferric acetate-uranium acetate reagent provides a simpler and quicker method. It does not suffer from interference due to the presence of Mn2+ ions and gives results comparable to the o-phthalaldehyde method and those using ultracentrifugation as the separation procedure.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Spectral control of metal admixtures in tetracycline]

Analysis of circular dichroism spectra made it possible to offer a method for estimation of tetracycline solutions contamination with metal ions. By its sensitivity the method is much superior to the spectrophotometric one used at present for determination of the antibiotic purity. In the latter method formation of complexes with metals is traced by batochromic displacement of the absorption spectra. The new method is rapid, relatively selective and requires comparatively small quantities of the substance for the analysis, which provides its use under both laboratory and manufacture conditions. The method is based on identification of the circular dichroism spectra of tetracycline complexes with metals in the long wavelength region. The presence of the circular dichroism concervative bands with strictly defined extremums in the spectra of tetracycline low acid solutions contaminated by multiply charged metal ions allowed vs. the circular dichroism spectra of pure tetracycline sample to conclude that the solution contained admixtures and to suggest their nature. It was shown that the charge, ion radius and tetracycline:metal relation were the factors defining the mark and location of the dichroism band extremums. At lambda(extr)-410-415 nm the tetracycline complexes with light metal ions such as Mg2+, Al3+ and Ca2+ were detected by the circular dichroism negative band in the spectra, while the complexes with heavy metal ions such as Sc3+, Sr3+, Cu3+, Cd3+, Ba2+, Y3+ and the cerium subgroup lanthanides were detected by the circular dichroism positive band. The tetracycline complexes with the lanthanides of the second half of the yttrium subgroup (Ho(3+)-Lu3+) were characterized by the presence of the circular dichroism minimum at lambda(min)-425 nm. When the tetracycline concentration was above 1.5 x 10(-3) M, multiligand complexes with circular dichroism negative extremum at lambda(min)-400 nm formed.     

Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the lactone and carboxylate forms of topotecan in human plasma

A selective and highly sensitive isocratic high performance liquid chromatographic (HPLC) method is described for simultaneous determination of lactone and carboxylate species of topotecan, in plasma. The method utilizes a protein precipitation step with cold methanol (-20 degrees C) for sample preparation followed by separation on a Novapack C(18) column using ammonium acetate buffer, acetonitrile and triethylamine (84:16:1.5, v/v) containing tetrabutyl ammonium hydrogen sulfate (TBAHS) (2 mM) with a pH of 5 as the mobile phase. The eluted peaks were detected by a fluorescence detector was set at an excitation wavelength of 380 nm and an emission wavelength of 527 nm. The method was validated in the range of lactone and carboxylate forms of topotecan concentrations from 0.05 to 75 ng/ml. Intra- and inter-day precision expressed by the relative standard deviation was less than 8.50% and inaccuracy did not exceed 10% for lactone and carboxylate forms of topotecan. The limit of quantitation was 0.05 ng/ml using 0.50 ml plasma. Stability studies in plasma and plasma extract indicated that topotecan is stable for at least 2 weeks at -70 degrees C.     

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

A new process for the simultaneous enzymic synthesis and purification of N-(benzyloxycarbonyl)- -aspartyl- -phenylalanine methyl ester (ZAPM), a precursor of aspartame, has been developed. The enzymic reaction between N-(benzyloxycarbonyl)- -aspartic acid (ZA) and -phenylalanine methyl ester (PM) was carried out in a biphasic hollow-fibre rector with an aqueous phase an a butyl acetate phase. The reaction took place in the aqueous phase and by maintaining the pH at 5, the product (ZAPM) was extracted into the organic phase. Product purity was greater than 90% and reasonable productivity could be achieved with this system.     

Biofiltration of Butyl Acetate and Xylene Mixtures Using a Trickle‐Bed Air Biofilter

Butyl acetate and xylene mixtures are commonly encountered from the manufacture of semi‐conductor or opto‐electronic apparatuses. The release of these substances into the ambient air may have a negative effect on the air quality. This study attempts to employ a trickle‐bed air biofilter for treating butyl acetate and xylene mixtures under different gas flow rates and influent concentrations. Almost complete VOC removal could be attained with influent carbon loadings of BA (butyl acetate) and X (xylene) below 40 and 15 g/m³h, respectively. As the influent carbon loadings of BA and X were increased up to 150 and 110 g/m³h, removal efficiencies higher than 80 % were achieved. Therefore, the trickle‐bed air biofilter (TBAB) appeared efficient in the control of emissions containing mixtures of butyl acetate and xylene with low to medium carbon loadings. The removal efficiencies of butyl acetate were higher than those of xylene, indicating that butyl acetate was the substrate preferred in the utilization of butyl acetate and xylene mixtures by the microorganisms. Carbon recoveries of 98–101 % were achieved, demonstrating the accuracy of results. The carbon mass rate of the liquid effluent was approximately two to three orders of magnitude less than that of the CO₂ effluent, indicating that the dissolved VOCs and their derivatives in the leachate were present in a negligible amount in the reactor. Applicable operating conditions of the TBAB unit for treating BA and X mixtures were suggested.     

高效液相色谱法测定重组人干扰素α-2b注射液中乙二胺四乙酸二钠含量

目的:利用高效液相色谱(HPLC)法测定重组人干扰素α-2b注射液中EDTA二钠(乙二胺四乙酸二钠)的含量。方法:将EDTA二钠与氯化铁溶液于70℃水浴中反应20 min左右;色谱柱为SunFire C18(250 mm×4.6 mm,5μm,Waters),流动相为5%甲醇+95%0.64 g/L四丁基溴化铵和4.1 g/L三水合乙酸钠混合液,用冰乙酸调pH值至4.0,流速1 mL/min,检测波长254 nm。结果:该测定方法线性范围为0.025~0.5 g/L,线性关系良好(r=0.9997),加样回收率为98.27%(n=9,RSD=2.55%)。结论:本方法准确、快速、可靠,可用于重组人干扰素α-2b注射液中EDTA二钠含量的测定。     

A rapid method for extraction of zearalenone and zeraralenols in fermented corn

A rapid extraction method is described for isolation of zearalenone and α- and β-trans-zearalenols from laboratory fermented corn. Corn fermented withFusarium crookwellense at 25°C for 2 weeks was agitated for 5 minutes in acetone. The acetone extract was evaporated to dryness and the remaining residue was chromatographed on a silica gel column with hexane:ethyl acetate (8:2). The products eluted with the hexane:ethyl acetate mixture in 1–1/2 column volumes and were identified by regular phase and C-18 reverse-phase thin layer chromatography. The products were verified by gas chromatography-mass spectroscopy. Product recoveries were 62.5–70% for the zearalenols and 70% for zearalenone in the range 0.5–50 mg/Kg.