Determination of bulleyaconitine A in human plasma by liquid chromatography-electrospray ionization tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization tandem mass spectrometry (MS-MS) was developed for the determination of bulleyaconitine A (BLA) in human plasma. BLA and internal standard (I.S.) ketoconazole were extracted from the plasma by a liquid-liquid extraction. The supernatant was evaporated to complete dryness and reconstituted with acetonitrile containing 0.1% acetic acid before injecting into an ODS MS column. The gradient mobile phase was composed of a mixture of acetonitrile (containing 0.1% acetic acid, v/v) and 0.1% acetic acid aqueous solution eluted at 0.3 ml/min. BLA and I.S. were determined by multiple reaction monitoring using precursor-->product ion combinations at m/z 644.6-->584.3 and 531.2-->81.6, respectively. Linearity was established for the concentration range of 0.12-6 ng/ml. The recoveries of BLA ranged from 96.93 to 113.9% and the R.S.D. was within 20%. The method is rapid and applicable to the pharmacokinetic studies of BLA in human.     

The uptake of [14C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Rabbit ear cartilage was incubated with [¹⁴C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.     

The uptake of [C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Rabbit ear cartilage was incubated with [¹⁴C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.     

A rapid assay for activity of phospholipase A2 using radioactive substrate

A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.     

A Method for the Selective Removal of Deoxyribonucleic Acid from Tissue Sections

The deoxyrihonucleic acid (DNA) of chromatin undergoar depurinization on mild acid hydrolysis with a picric acid-formaldehyde mixture (Bouin's fluid). The apurinic acid thus formed is degraded by condensation with aniline and is lost from tissue sections, but ribonucleic acid (RNA) in nucleoli and cytoplasm is well preserved. Technique: Fi in Carnoy's fluid (ethanol:acetic acid 3:1 or ethanol:chloroform:acetic acid 6:3:1) or in aldehydes (10% formalin or 2.5% glutaraldehyde bsered to pH 7.0). Hydrolyse deparaEnii sections 12-24 hr at 27-50 C in Bouin's fluid, wash in distilled water, immerse in 25% (v/v) acetic acid, treat 1 hr at 27-30 C with 10% (v/v) dine in 25% acetic acid, wash in 25% acetic acid and then in water. Stain 10-40 min with 0₃% toluidine blue in 0.05 M potassium biphthalate bder (pH 4.0); rinse in distilled water, pass to 10% (w/v) ammonium molybdate for 1 min, rinse again in water and pass through tert-butanol and xylene to a synthetic resin. Chromatin and chromosomes are pale green; RNA in nucleoli and cytoplasm deep purple.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Determination of nicotine and cotinine in tobacco harvesters' urine by solid-phase extraction and liquid chromatography

A solid-phase extraction method using Drug Test-1 column containing chemically modified silica as a solid support for sample clean up and reversed phase ion-paired high-pressure liquid chromatography method have been developed for the simultaneous determination of nicotine and its metabolite cotinine from the urine samples. Mobile phase was consisted of acetate buffer (containing 0.03 M sodium acetate and 0.1 M acetic acid) pH 3.1 and acetonitrile (78:22% (v/v)) containing 0.02 M sodium octanosulfonate as an ion pair agent. pH of the mobile phase was adjusted to 3.6 with triethylamine for better resolution and to prevent peak tailing. The linearity was obtained in the range of 0.5-10 microg/ml concentrations of nicotine and cotinine standards. The correlation coefficients were 0.998 for cotinine and 0.999 for nicotine. The recoveries were obtained in the range of 79-97% with average value of 85% for nicotine and in the range of 82-98% with average value of 88% for cotinine. The limit of detection was 2 ng/ml for cotinine and 5 ng/ml for nicotine with 2 ml urine for extraction, calculated by taking signal to noise ratio 10:3. The intra-day co-efficient of variation (CV) were <4 and 7% and inter-day CV were <9 and 7% for nicotine and cotinine, respectively. The method was applied to the urine samples of tobacco harvesters, who suffer from green tobacco sickness (GTS) to check the absorption of nicotine through dermal route during the various processes of tobacco cultivation due to its good reproducibility and sensitivity.     

ADP ribosylation of rat liver lysine-rich histone in vitro.

Purified rat liver nuclei were incubated with [14C]-NAD+ and the various nuclear protein fractions were separated. Forty per cent of the total radioactivity incorporated was associated with the histone fraction. Of this, about 50% was extracted with H1, in 0.5 N perchloric acid. When crude H1 was purified and fractionated into five different subfractions by chromatography on Bio-Rex 70, it was found that all the H1 subfractions contained radioactivity. This radioactive material was identified as oligomers of adenosine diphosphate ribose (ADP-Rib) with an average chain length which corresponded to trimers. The extent of the modification was dependent on the concentration of NAD+. About 60% of the H1 molecules were modified with a concentration of 1 mM NAD+. The presence of these oligomers of ADP-Rib introduced a large degree of microheterogeneity to H1 as detected by electrophoresis in polyacrylamide gels containing 2.5 M urea and 0.9 N acetic acid. Bands of H1 with 10 to 20% less mobility than the unmodified H1 were present. Also, as a consequence of large content of ADP-Rib, the absorption maximum shifted from 275 to 259 nm. The half-life of the bond between the oligomers of ADP-Rib and H1 was about 3 min at 37 degrees C in the presence of 0.1 N NaOH, and 10 m1 were modified. The site of ADP ribosylation in the NH2-terminal half was localized in the tryptic peptide extending from the NH2-terminal end to lysine 15. The site of modification of the COOH-erminal half was localized in the tryptic peptide which contained the only glutamic acid residue in this fragment of H1...     

Biosynthesis of [3H]7 alpha-hydroxy-, 7 beta-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [(3)H]-labeled 7 alpha-hydroxy-DHEA (7 alpha-OH-DHEA), 7 beta-hydroxy-DHEA (7 beta-OH-DHEA), and 7-oxo-DHEA substrates from 50 microM [1,2,6,7-(3)H]DHEA (specific radioactivity 60-80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography-mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7 alpha-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2h/2 ml, respectively; 82% conversion), while in the presence of 1mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)), more 7-oxo-DHEA than 7 alpha-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed. When longer reaction times were used with NADPH and NADP(+), a mixture of 7 alpha-OH-DHEA, 7 beta-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.     

Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetate: isooctane: glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 v/v for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 μg of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.     

Cell-free synthesis of mouse corticotropin. Evidence for two high molecular weight gene products.

Polysomes or mRNA prepared from cultured AtT-20/D16v mouse pituitary adenocarcinoma cells direct the efficient incorporation of amino acid into newly synthesized material in the presence of wheat germ translational factors. A significant franction of the total cell-free product is specifically immunoprecipitable with corticotropin antibody purified by immune affinity chromatography. Analysis of the cell-free synthesized immunoreactive products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that two high molecular weight corticotropin species (Mr congruent to 32,500 and 28,000) are synthesized in an approximate 2:1 ratio. Neither product contains carbohydrate based upon concanavalin A chromatography or exposure to polysaccharidases. The smaller molecular weight product does not appear to arise from proteolytic processing since both species are synthesized in approximately the same ratio in cell-free reaction mixtures directed by either polysomes or mRNA. These results suggest that AtT-20/D16v cells contain two distinct mRNA poluations specifying the synthesis of two different high molecular weight forms of mouse corticotropin.     

Peroxynitrite and NO donors form colored nitrite adducts with sinapinic acid: potential applications

Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid, SA) reacted with peroxynitrous acid at neutral pH with a second-order rate constant of 812 M(-1)s(-1), to yield a red product (lambda(max), 532 nm). The identical colored product could be formed with acidified decomposed peroxynitrous acid solutions or nitrite at slower rates (0.1M HCl, 8.32 M(-1)s(-1); 10% acetic acid, 0.0004 M(-1)s(-1)). The red compound is thought to be O-nitrososinapinic acid (3,5-dimethoxy-4-nitrosooxycinnamic acid) which can be formed by reaction with either peroxynitrous acid or nitrous acid. The extinction coefficient of O-nitrososinapinic acid (ONSA) was estimated to be 8419 M(-1)cm(-1) at 510 nm in 10% acetic acid and 90% acetonitrile. ONSA was also formed via NO(+) transfer from S-nitrosoglutathione (GSNO). ONSA in turn can S-nitrosate low molecular weight thiols and protein thiols. SA was also shown to act as a peroxynitrite sink as it effectively prevented the oxidation of dihydrorhodamine under physiological conditions. The fact that O-nitrososinapinic acid is stable and can be used to S-nitrosate thiol containing amino acids, peptides, and proteins makes it a potentially useful reagent in the study of S-nitrosothiol biochemistry and physiology. In addition, the relatively high extinction coefficient of O-nitrososinapinic acid means that it could be utilized as an analyte for the spectroscopic detection of peroxynitrite or NO(+)-donors in the submicromolar range.     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Hydration of oleic acid by a Selenomonas strain from the ovine rumen

J.A. HUDSON, C.A.M. MACKENZIE AND K.N. JOBLIN. 1996. A Selenomonas sp., isolated from the ovine rumen, was characterized with regard to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydration occurred only in stationary phase in a medium containing 0.1%, 0.5% (w/v) galactose or 0.5% (w/v) glucose, but not in a medium containing 1% galactose. Growth under a hydrogen headspace did not result in the production of stearic acid, the biohydrogenated product of oleic acid. Linoleic and linolenic acids (0.1% v/v) were not hydrated. It is concluded that the growing bacterium is unlikely to contribute to oleic acid hydration in the rumen.     

Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.     

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.     

The effect of insulin on amino acid metabolism and glycogen content in isolated liver cells of juvenile coho salmon, Oncorhynchus kisutch

Amino acid transport and [14C]leucine incorporation into liver proteins as well as the secretion of proteins into incubation medium were studied in liver cells isolated from coho salmon (Oncorhynchus kisutch) parr. Pink salmon (Oncorhynchus gorbuscha) or mammalian (bovine) insulin caused a significant increase in TCA-precipitable radioactivity from both cells and incubation medium. The effects appeared at insulin concentration of 10(-8) M with a maximal response at 5 X 10(-8) M. The radioactivity of the TCA-soluble fraction was not changed by insulin. Insulin increased the amount of the non-metabolized amino acid [14C]cycloleucine, in the TCA-soluble fraction of hepatocytes. The glycogen content of hepatocytes was increased in the presence of insulin at 10(-9) M but was not changed from the control value in the presence of insulin at 10(-8) M.     

Factors affecting the formation of 10-hydroxystearic acid from oleic acid by a ruminal strain of Enterococcus faecalis

 A ruminal strain of Enterococcus faecalis was characterised with respect to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydroxy fatty acid was produced after growth had ceased and the carbon source was almost exhausted. Hydroxy fatty acid production was equally rapid whether the inoculum had been grown in the presence of oleic acid or not, and almost complete conversion was achieved when oleic acid was present at a concentration of up to 0.5% (v/v). Incubation under a hydrogen headspace did not result in biohydrogenation of oleic acid. In pH-controlled batch culture the proportion of oleic acid hydrated varied with the pH of incubation, with more hydration at lower pH. Growth was retarded in the presence of 0.1% (v/v) linoleic acid, inhibited by the same concentration of linolenic acid and did not result in the formation of hydrated products from these substrates. If this organism is able to transform oleic acid in the rumen then the only product likely to be formed is 10hydroxystearic acid. Received: 17 July 1995/Received last revision: 24 October 1995/Accepted: 30 October 1995