A radioimmuno—chromatographic scanning method for the analysis of testosterone conjugates in urine and serum

A method for the analysis of testosterone (and 5α-dihydrotestosterone) conjugates in human serum and urine samples is described. The samples were brought to pH 1 and extracted with a diethyl ether—methanol mixture. After evaporation the residues were run in a thin-layer chromatography system, individual samples' paths were cut into 1-cm long pieces and eluted with methanol. The methanol was evaporated and the residue subjected to acid hydrolysis. The released steroid was extracted by diethyl ether and measured by radioimmunoassay. The methodology described represents a new approach to the qualitative and quantitative study of steroid conjugates in serum and urine, and can easily be applied to the study of steroid conjugates in other biological material.     

Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.     

Expression of glycosylated haloalkane dehalogenase LinB in Pichia pastoris

Heterologous expression of the bacterial enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 in methylotrophic yeast Pichia pastoris is reported. The haloalkane dehalogenase gene linB was subcloned into the pPICZalphaA vector and integrated into the genome of P. pastoris. The recombinant LinB secreted from the yeast was purified to homogeneity and biochemically characterized. The deglycosylation experiment and mass spectrometry measurements showed that the recombinant LinB expressed in P. pastoris is glycosylated with a 2.8 kDa size of high mannose core. The specific activity of the glycosylated LinB was 15.6 +/- 3.7 micromol/min/mg of protein with 1,2-dibromoethane and 1.86 +/- 0.36 micromol/min/mg of protein with 1-chlorobutane. Activity and solution structure of the protein produced in P. pastoris is comparable with that of recombinant LinB expressed in Escherichia coli. The melting temperature determined by the circular dichroism (41.7+/-0.3 degrees C for LinB expressed in P. pastoris and 41.8 +/- 0.3 degrees C expressed in E. coli) and thermal stability measured by specific activity to 1-chlorobutane were also similar for two enzymes. Our results show that LinB can be extracellularly expressed in eukaryotic cell and glycosylation had no effect on activity, protein fold and thermal stability of LinB.     

Exploring the structure and activity of haloalkane dehalogenase from Sphingomonas paucimobilis UT26: evidence for product- and water-mediated inhibition

The hydrolysis of haloalkanes to their corresponding alcohols and inorganic halides is catalyzed by alpha/beta-hydrolases called haloalkane dehalogenases. The study of haloalkane dehalogenases is vital for the development of these enzymes if they are to be utilized for bioremediation of organohalide-contaminated industrial waste. We report the kinetic and structural analysis of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) in complex with each of 1,2-dichloroethane and 1,2-dichloropropane and the reaction product of 1-chlorobutane turnover. Activity studies showed very weak but detectable activity of LinB with 1,2-dichloroethane [0.012 nmol s(-1) (mg of enzyme)(-1)] and 1,2-dichloropropane [0.027 nmol s(-1) (mg of enzyme)(-1)]. These activities are much weaker compared, for example, to the activity of LinB with 1-chlorobutane [68.2 nmol s(-1) (mg of enzyme)(-1)]. Inhibition analysis reveals that both 1,2-dichloroethane and 1,2-dichloropropane act as simple competitive inhibitors of the substrate 1-chlorobutane and that 1,2-dichloroethane binds to LinB with lower affinity than 1,2-dichloropropane. Docking calculations on the enzyme in the absence of active site water molecules and halide ions confirm that these compounds could bind productively. However, when these moieties were included in the calculations, they bound in a manner similar to that observed in the crystal structure. These data provide an explanation for the low activity of LinB with small, chlorinated alkanes and show the importance of active site water molecules and reaction products in molecular docking.     

Determination of alpha-bisabolol in human blood by micro-HPLC-ion trap MS and head space-GC-MS methods

Alpha-bisabolol is a compound present in some essential oils, widely distributed in several plants, including camomile. Two different methods for analysing an essential oil, such as alpha-bisabolol in human blood are reported: the first uses micro-liquid chromatography-electrospray ionisation-mass spectrometry (muHPLC-ESI-MS), whereas the second is based on "head space" injection coupled to gas chromatography-mass spectrometry (HS-GC-MS). For LC-ESI-MS, human blood samples, spiked with alpha-bisabolol, were extracted with hexane and evaporated to dryness under air stream. The residue was then reconstituted with methanol and injected into a C18 column, connected to an ion trap mass spectrometer equipped with an ESI source. Spectra were recorded in the positive ion, selected ion monitoring mode. The detection limit of alpha-bisabolol in blood was 0.125 micromol/l. The preparation of samples for the analysis in HS-GC-MS was limited to blood dilution with water (0.5 ml blood + 1 ml water). Head space vials were heated at 125 degrees C for 1 h before automatic injection. The HS-GC-MS detection limit (0.13 micromol/l) was similar to that achieved with the muHPLC-ESI-MS method. Successful tests were performed to verify if alpha-bisabolol could be directly measured by the HS-GC-MS method in different biological samples (blood, urine, faeces, homogenate tissues) from rats treated with the camomile essential oil.     

Determination of sotalol in human cardiac tissue by high-performance liquid chromatography

A sensitive and quantitative reversed-phase HPLC method for the analysis of -sotalol in human atria, ventricles, blood and plasma was developed. Sotalol was determined in about 100 mg of human right atria, left ventricles, and in 500 μl of blood and plasma samples of patients undergoing coronary bypass surgery or heart transplantation. Patients were taking 80–160 mg of sotalol as an antiarrhythmic agent. Atenolol was used as an internal standard certifying high precision of measurement. Sotalol blood and plasma concentrations correlated linearly to the obtained signals from 26.5 ng/ml to 2.12 μg/ml. Sotalol tissue concentrations showed linearity between 0.27 ng/mg and 10.6 ng/mg wet weight. The limit of quantitation was 0.27 ng/mg at a signal-to-noise ratio of 10. Sotalol was extracted from homogenized tissue with a buffer solution (pH 9) and the remaining pellet was extracted with methanol. The methanol extract was evaporated under nitrogen and reconstituted in buffer (pH 3). The whole extract was cleaned by solid-phase column extraction, eluted with methanol, evaporated again, reconstituted in the mobile phase (acetonitrile-15 mM potassium phosphate buffer pH 3, 17:83, v/v) and injected onto the HPLC column (Spherisorb C₆ column, 5 μm,, 150×4.6 mm I.D). For the detection of sotalol, the UV wavelength was set to 230 nm. Recoveries of sotalol and atenolol in atria and ventricles were 65.6 and 75.0%, respectively. Intra- and inter-assay coefficients of variation for tissue concentrations were 3.38 and 6.14%, respectively. Intra- and inter-assay accuracy for determined tissue sotalol concentrations were 94.9±6.3 and 99.6±4.1%.     

Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method

Methods were developed for the separation and measurement of lipid and protein hydroperoxides, which can be used for biological materials. Lipids were extracted with methanol:chloroform and their hydroperoxides measured in solutions of methanol and chloroform containing 110mM perchloric acid, xylenol orange, and ferrous iron. Proteins were isolated by precipitation with 0.2M perchloric acid. The precipitates were redissolved in 6M guanidine hydrochloride and washed with chloroform, and the hydroperoxides were measured in the presence of perchloric acid, xylenol orange, and ferrous iron. Optimum conditions for hydroperoxide measurements were established and the assays were applied to oxidized human blood serum and to cultured cells.     

Determination of monensin, salinomycin and narasin in muscle, liver and eggs from domestic fowl using liquid chromatography-electrospray mass spectrometry

A method is presented for the simultaneous determination of the three ionophores, monensin, salinomycin and narasin in muscle, liver and eggs from domestic fowl. Samples are homogenised in methanol and any ionophores present are extracted into toluene-hexane. The extracts are concentrated and injected into a bench-top electrospray LC-MS system. Chromatography is carried out using an end-capped reversed-phase column, with a mobile phase consisting a mixture of acetonitrile, methanol, tetrahydrofuran, trifluoroacetic acid and water. Using single-ion monitoring, the ionophores can be detected down to the 1 ng/g level.     

Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids

Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reversephase chromatography on cartridges containing a C₁₈ spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis.     

Determination of [d-Ala2, d-Leu5]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Solubility of the intramitochondrially synthesized protein and other membrane proteins of rat liver mitochondria in acidic or neutral chloroform-methanol

Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, $\text{v}\text{v}$/, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver $\text{in vivo}$ is presented.     

Nonconventional hydrolytic dehalogenation of 1-chlorobutane by dehydrated bacteria in a continuous solid-gas biofilter

Rhodococcus erythropolis NCIMB 13064 and Xanthobacter autotrophicus GJ10 are able to catalyze the conversion of halogenated hydrocarbons to their corresponding alcohols. These strains are attractive biocatalysts for gas phase remediation of polluted gaseous effluents because of their complementary specificity for short or medium and for mono-, di-, or trisubstituted halogenated hydrocarbons (C2-C8 for Rhodococcus erythropolis and C1-C4 for Xanthobacter autotrophicus).After dehydration, these bacteria can catalyze the hydrolytic dehalogenation of 1-chlorobutane in a nonconventional gas phase system under a controlled water thermodynamic activity (a(w)). This process makes it possible to avoid the problems of solubility and bacterial development due to the presence of water in the traditional biofilters.In the aqueous phase, the dehalogenase activity of Rhodococcus erythropolis is less sensitive to thermal denaturation and the apparent Michaelis-Menten constants at 30 degrees C were 0.4 mM and 2.40 micromol min(-1) g(-1) for Km and Vmax, respectively. For Xanthobacter autotrophicus they were 2.8 mM and 0.35 micromol min(-1) g(-1). In the gas phase, the behavior of dehydrated Xanthobacter autotrophicus cells is different from that observed with Rhododcoccus erythropolis cells. The stability of the dehalogenase activity is markedly lower. It is shown that the HCl produced during the reaction is responsible for this low stability. Contrary to Rhodococcus erythropolis cells, disruption of cell walls does not increase the stability of the dehalogenase activity.The activity and stability of lyophilized Xanthobacter autotrophicus GJ10 cells are dependant on various parameters. Optimal dehalogenase activity was determined for water thermodynamic activity (a(w)) of 0.85. A temperature of 30 degrees C offers the best compromise between activity and stability. The pH control before dehydration plays a role in the ionization state of the dehalogenase in the cells. The apparent Michaelis-Menten constants Km and Vmax for the dehydrated Xanthobacter autotrophicus cells were 0.07 (1-chlorobutane thermodynamic activity) and 0.08 micromol min(-1) g(-1) of cells, respectively. A maximal transformation capacity of 1.4 g of 1-chlorobutane per day was finally obtained using 1g of lyophilized Xanthobacter autotrophicus GJ10 cells.     

The use of non-aqueous chloroform/methanol extraction for the delipidation of brain with minimal loss of enzyme activities.

When freeze-dried brain was extracted at -4-0degrees C with dry chloroform/methanol (2:1 v/v), four of the five enzymes examined were recovered in the diethyl ether-washed residue without inactivation. By contrast, extraction with chloroform/methanol (2:1 v/v) in the presence of water destroyed activities of all the enzymes examined. The amounts of major lipids extracted were similar whether extraction was done in the absence or presence of water. The study was carried out with special interest in 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37), which is firmly bound to the membrane structures of brain white matter.     

The acyl lipid composition of wheat leaves and moss protonemata using a new, non-carcinogenic extraction solvent system

As chloroform has proved to be carcinogenic we were looking for an alternative solvent system for chloroform:methanol widely used in plant lipid investigations. The lipids from leaves of wheat ( Triticum aestivum L. cv. Vakka) and from protonemata of the moss Ceratodon purpureus (Hedw.) Brid. were extracted with two petroleum ether:methanol solvent systems. The polar lipids were separated by two-dimensional thin-layer chromatography and the amounts of each lipid class were compared with those obtained from chloroform:methanol (2:1, v/v) extractions. The significantly higher amounts of phosphatidylinositol observed in petroleum ether:methanol (1:1, v/v) extraction suggest that the small amounts reported earlier in plants may be an artefact relating to the solvent system used. As petroleum ether:methanol (1:1, v/v) proved to be at least as good a solvent system as chloroform:methanol (2:1, v/v) we propose it as an alternative extractant for plant polar lipids.     

Validated high-performance liquid chromatographic assay for simultaneous determination of dacarbazine and the plasma metabolites 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide

Dacarbazine (DTIC) is a prodrug that is clinically effective in the treatment of Hodgkin’s disease, melanoma and soft tissue sarcoma. To better characterize the clinical pharmacology of parent drug and reactive metabolites, a reversed-phase HPLC method with UV detection was developed for simultaneous determination of dacarbazine and the metabolites 5-(3-hydroxymethyl-3-methyl-1-triazeno)imidazole-4-carboxamide (HMMTIC) and 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC). Chromatographic separation was achieved with a Zorbax SB-CN column and with a mobile phase of 80% 50 mM ammonium phosphate, pH 6.5, 20% methanol and 0.1% triethylamine. HMMTIC, MTIC and DTIC were extracted from plasma with methanol precipitation of the proteins. Recovery of DTIC and the metabolites from whole blood was greater than 92%. Rapid processing of whole blood, methanol extraction and storage at −70°C substantially increased the stability of HMMTIC and MTIC from less than 15 min to 3 days. Precision for HMMTIC, MTIC and DTIC ranged from 3.7 to 16.3% relative standard deviation. The accuracy ranged from 101 to 114% for all three analytes. The validated assay was used to determine the pharmacokinetic data for dacarbazine and its active metabolites for human patients with recurrent glioma receiving DTIC intravenously.     

Preparation of biological fluids for simultaneous analysis of prostaglandin cyclo-oxygenase synthesized compounds by gas chromatography with electron capture detection

A method for isolating climax products of the arachidonic acid cascade from biological fluids is described which allows simultaneous measurement of PGs (PGE2, PGD2, 6-keto-PGF1 alpha, TXB2, 6-keto-PGE1, 6, 15-diketo-13,14-dihydro-PGF1 alpha) by electron capture detection of pentafluorobenzyloxime methyl ester trimethylsilyl (TMS) ether derivatives. PGs are adsorbed onto Amberlite XAD-2 from acidified solution and nonadhering substances removed by sequential administration of water, then petroleum ether. PGs are extracted into methanol. Following evaporation and reconstitution in water, the PGs are extracted into ethyl acetate at pH 3 and the ethyl acetate extracts are purified by lipidex column chromatography. Derivatization to pentafluorobenzyloxime methyl ester TMS ethers of PGs in the sample is followed by separation on either glass packed-columns or SCOT capillary columns, and detection by an electron capture detector. PGA2, added to the unpurified sample, is used as an internal standard for quantification. The methods have performed well on all biological fluids thus far examined. Examples of chromatographs from urine, Krebs-perfused lung effluents, and blood are shown.     

Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies

A reversed phase HPLC-MS/MS method has been developed and validated for the quantitative bioanalysis of acetaminophen in dried blood spots (DBS) prepared from small volumes (15 microL) of dog blood. Samples were extracted for analysis with methanol. Detection was by positive ion TurboIonSpray ionisation combined with selected reaction monitoring MS. The analytical concentration range was 0.1-50 microg/mL. The intra-day precision and bias values were both less than 15%. Acetaminophen was stable in DBS stored at room temperature for at least 10 days. The methodology was applied in a toxicokinetic (TK) study where the data obtained from DBS samples was physiologically comparable with results from duplicate blood samples (diluted 1:1 (v/v) with water) analysed using identical HPLC-MS/MS conditions. This work demonstrates that quantitative analysis of a drug extracted from DBS can provide high quality TK data while minimising the volume of blood withdrawn from experimental animals, to an order of magnitude lower than is current practice in the pharmaceutical industry. This is the first reported application of DBS analysis to a TK study in support of a safety assessment study. The success of this and similar, related studies has led to the intent to apply DBS technology as the recommended analytical approach for the assessment of pharmacokinetics (PK)/TK for all new oral small molecule drug candidates, which have previously demonstrated a successful bioanalytical validation.     

Antihypertensive activities of a solid-state culture of Taiwanofungus camphoratus (Chang-chih) in spontaneously hypertensive rats

Wild and solid-state cultures (SSC) of Taiwanofungus camphoratus (aka Antrodia camphorata and Chang-chih [CC]) were sequentially extracted with cold water, methanol, and hot water to get cold-water-soluble (CWS), methanol-soluble (MS), and hot-water-soluble (HWS) extracts, respectively. Only the MS extract exhibited angiotensin-converting enzyme (ACE) inhibitory activities. The antihypertensive effects of the MS extract (10 mg/kg BW) were measured in spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats. MS extract of the SSC type was able to effectively lower the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHR, but not of WKY rats, the results being significantly different from those for distilled water only (the blank). However, wild CC and its MS extract were not as effective as the SSC type in reducing SHR blood pressure and had no effect on WKY rats. SSC-type CC might be developed into a health food with the ability to regulate blood pressure.     

Acetylcholine-binding substance extracted by using organic solvent and acetylcholine receptor of electric organ of Narke japonica.

1. Proteolipid was extracted from the electric organ of Narke japonica by using chloroform/methanol (2:1, v/v). This extract was separated into acetylcholine-binding and non-binding substances by column chromatography. However, acetylcholine-binding substances did not show the characteristic properties of protein. 2. The membrane fragments of the electric organ were separated into three main parts by sucrose density gradient centrifugation. From the heaviest, the fractions were acetylcholine receptor rich, ATPase rich, and acetylcholinesterase rich. 3. The membrane fraction having acetylcholine receptor showed the excitability, the increase of Na+ permeability by the application of cholinergic agonists. However, the acetylcholine binding substance extracted by the organic solvent was richer in the lighter fraction. This substance differed from the true acetylcholine receptor.