Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells

A sensitive, nonradioactive analytical method has been developed to simultaneously determine the concentrations of farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) in cultured cells. Following extraction, enzyme assays involving recombinant farnesyl protein transferase or geranylgeranyl protein transferase I are performed to conjugate FPP or GGPP to dansylated peptides. The reaction products are then separated and quantified by high-performance liquid chromatography coupled to a fluorescence detector at the excitation wavelength 335 nm and the emission wavelength 528 nm. The retention times for farnesyl-peptide and geranylgeranyl-peptide are 8.4 and 16.9 min, respectively. The lower limit of detection is 5 pg of FPP or GGPP ( approximately 0.01 pmol). A linear response has been established over a range of 5-1000 pg ( approximately 0.01-2 pmol) with good reproducibility. The method has been used to determine the levels of FPP (0.125+/-0.010 pmol/10(6)cells) and GGPP (0.145+/-0.008 pmol/10(6)cells) in NIH3T3 cells. Furthermore, changes in FPP and GGPP levels following treatment of cells with isoprenoid biosynthetic pathway inhibitors were measured. This method is suitable for the determination of the concentrations of FPP and GGPP in any cell type or tissue.     

Simultaneous determination of catecholamines and unconjugated 3,4-dihydroxyphenylacetic acid in brain tissue by ion-pairing reverse-phase high-performance liquid chromatography with electrochemical detection

A rapid, sensitive method was developed for the simultaneous assay of catecholamines and 3,4-dihydroxyphenylacetic acid in rat brain tissue. The method is simple, involving only tissue disruption, adsorption of the catechols onto alumina, desorption, and injection into a reverse-phase high-performance liquid chromatography system. Selectivity and high sensitivity are obtained using electrochemical detection. The addition of 3,4-dihydroxyphenylacetic acid determination to assays for catecholamines allows one to observe effects of pharmacological maniqulations on in vivo monoamine oxidase activity and/or turnover of dopamine as well as effects on catecholamine concentrations.     

High-performance liquid chromatographic determination of catecholamines and their precursor and metabolites in human urine and plasma by postcolumn derivatization involving chemical oxidation followed by fluorescence reaction.

A high-performance liquid chromatographic method for the determination of catecholamines and their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine, and L-DOPA), acidic compounds (3,4-dihydroxyphenylacetic acid, vanillyl-mandelic acid, and homovanillic acid), and alcoholic compound [4-hydroxy-3-methoxyphenyl)ethylene glycol)] in human urine and plasma. Urine and plasma samples deproteinized with perchloric acid in the presence of isoproterenol and 3,4-dihydroxyphenylpropanoic acid (internal standards) are fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction). The compounds in each fraction are separated by an ion-pair reversed-phase chromatography on a TSK gel ODS-80TM with isocratic elution and on-line derivatized by periodate oxidation followed by a fluorescence reaction using meso-1,2-diphenylethylenediamine. The detection limits (S/N = 5) vary from 0.5 to 95 pmol/ml, depending on the compounds.     

High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection

A high-performance liquid chromatography-fluorescence procedure for the determination of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, dopamine, and norepinephrine has been developed. The method uses an ion-pairing system on an Ultrasphere ODS (5-microns) column with detector wavelength settings of excitation at 290 nm and emission at 330 nm. The procedure has been used to quantitate these indoleamines and catecholamines in rat brain tissue after homogenization in a perchloric acid solution; an aliquot of this solution is injected directly onto the HPLC column. Column sensitivities range from 6.1 pmol for tryptophan to 1.1 pmol for 5-hydroxytryptamine.     

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92–98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.     

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10–10 000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17°C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Determination of a cysteine protease inhibitor and its ethyl ester in mouse serum and muscle by liquid chromatography—mass spectrometry

A liquid chromatographic—mass spectrometric method is described for the determination of a cysteine protease inhibitor (E64C) and its ethyl ester in mouse serum and muscle samples. The compounds in the sample, after deproteinization and solid-phase extraction, were separated by isocratic reversed-phase high-performance liquid chromatography and detected by on-line mass spectrometry. The use of an aqueous mobile phase containing methanol and 30 mM ammonium trichloroacetate provided abundant protonated molecular ions of the compounds in the atmospheric pressure chemical ionization interface of the detection system. The method permitted the quantitative determination of the inhibitors without internal standards in the biological matrices. The detections limits for the compounds, in the selected-ion monitoring mode, were 10–15 pmol on-column, at a signal-to-noise ratio of 5.     

Catecholamines and dihydroxyphenylalanine in metamorphosing larvae of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

The content of catecholamines and dihydroxyphenylalanine in larvae of the nudibranch Phestilla sibogae was analyzed by high-performance liquid chromatography with electrochemical detection. Dihydroxyphenylalanine, norepinephrine and dopamine were identified in larvae of all ages examined (5 through 12 days post-fertilization). Dihydroxyphenylalanine could be accurately quantified only in larvae of ages 8 through 12 days, when its average concentration increased from 0.62 to 6.71 × 10⁻² pmol μg protein⁻¹. Between ages 5 and 12 days dopamine rose from 0.081 to 0.616 pmol μg protein⁻¹, and norepinephrine from 0.45 to 2.17 × 10⁻² pmol μg protein⁻¹. Dihydroxyphenylalanine, dopamine and norepinephrine were also measured at different stages of metamorphic progress in 10- to 12-day larvae. Dihydroxyphenylalanine increased by a factor of 2.4 between the onset and completion of metamorphosis, but levels of dopamine and norepinephrine remained stable. One millimolar alpha-methyl-dl-m-tyrosine, an inhibitor of catecholamine synthesis, inhibited natural metamorphosis and depleted endogenous norepinephrine and especially dopamine, respectively, to 75% and 35% of control values. The existence of unexpectedly high levels of catecholamines in metamorphically competent larvae, and the association of catecholamine depletion with inhibition of metamorphosis, indicate that these compounds may participate in the control of gastropod development. Accepted: 18 April 1997     

Application of high-performance liquid chromatographic peptide purification to protein microsequencing by solid-phase Edman degradation

Peptide purification via high-performance liquid chromatography (HPLC) and solid-phase sequencing were integrated to form a system allowing the determination of complete sequence information on a microscale without the use of radiolabels or modified phenylisothiocyanate. Mixtures of peptides (500 pmol to 10 nmol) resulting from proteolytic digestion or chemical cleavage were applied directly to reverse-phase columns. The columns, equilibrated in either 10 mm KP_i or 0.05% trifluoroacetic acid, were then developed using acetonitrile gradients. Eluates were monitored nondestructively by direct ultraviolet detection at both 214 and 254 nm. Each peak was collected as a discrete fraction, and purity was assessed by amino acid analysis prior to covalent attachment to a solid support for sequence analysis. Activation of the peptide carboxyl terminus via a water soluble carbonyldiimide was the solid-phase coupling method used 90% of the time. Coupling yields averaged 52% of starting material. Sequence analysis was performed in the range 100 pmol to 4 nmol of coupled peptide. Phenylthiohydantoin-amino acids were identified by reverse-phase HPLC using ultraviolet detection.     

Determination of 17-oxosteroid glucuronides and sulfates in urine and serum by fluorescence high-performance liquid chromatography using dansyl hydrazine as a pre-labeling reagent

A fluorescence high-performance liquid chromatographic method is described for the direct determination of conjugated 17-oxosteroids in biological fluids without hydrolysis. Conjugated 17-oxosteroids are extracted with Sep-Pak C₁₈ cartridge, labeled with dansyl hydrazine in trichloroacetic acid—benzene solution and then separated by high-performance liquid chromatography on reversed-phase μBondapak C₁₈ column using 0.01M sodium acetate in methanol—water—acetic acid (65:35:1, v/v) as the mobile phase. The eluate is monitored by a fluorophotometer at 365 nm (excitation) and 520 nm (emission). Linearities of fluorescence intensities (peak heights) with the amounts of various conjugated 17-oxosteroids were obtained between 10 pmol and 100 pmol. This method is sensitive, reliable and useful for the simultaneous determination of conjugated 17-oxosteroids in urine and serum.     

Plasma catecholamines in resting rainbow trout,Salmo gairdneri Richardson,by high pressure liquid chromatography*

Plasma norepinephrine and epinephrine from cannulated trout were measured by high pressure liquid chromatography with electrochemical detection. The catecholamines were extracted with acid-washed alumina using a microfilter assembly which permitted analysis of small volumes of plasma. Mean (± S.D.) values for plasma norepinephrine and epinephrine were 1.83 (0.97) pmol ml^?1 and 8.95 (4.94) pmol ml^?1, respectively. These values are compared with catecholamine levels from other vertebrate species.     

Novel post-column derivatization method for the fluorimetric determination of norepinephrine and epinephrine

A novel method is described in which catecholamines are converted into fluorescent products by heating in alkaline borate buffer. The method was applied to the determination of norepinephrine and epinephrine after separation by high-performance liquid chromatography using a pellicular, strong cation exchanger. The new system is simpler than the system based on the trihydroxyindole reaction. It is suitable for the measurement of catecholamines in the range 0.25–20 ng. The assay of catecholamines in human urine is also described.     

Ca2+-stimulated catecholamine release from alpha-toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G proteins

Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in alpha-toxin-permeabilized cells with micromolar levels of free Ca2+. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca2+. Both types of experiments provide direct evidence for the operation of Ca2+-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca2+ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below 1 microM free Ca2+ and plateaus at 1-5 microM free Ca2+, while a second occurs in the presence of larger amounts of free Ca2+ (10-100 microM). Ca2+-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg2+/ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca2+-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.     

Degradation of dansyl polyamines on high-performance thin-layer chromatographic plates

Using high-performance thin-layer chromatography with in situ quantitation to measure dansylated polyamines in the range of 1–20 pmol, we found that dansylated polyamines apparently react with the silica gel of the plates. The fluorescence of the dansyl polyamines diminished with increase in the time interval between application of a sample to the plate and start of the chromatographic separation. Conversely, the fluorescence at the site of application increased with the length of the time interval, indicating the formation of polar reaction products. If this reaction is not accounted for, considerable errors in quantitation of dansyl polyamines may occur.     

High-performance liquid chromatographic assay for the simultaneous determination of sulfadoxine and pyrimethamine from whole blood dried onto filter paper

A method using solid-phase extraction and high-performance liquid chromatography is evaluated for the simultaneous determination of sulfadoxine and pyrimethamine from 0.1 ml of whole blood dried onto filter paper. Extraction recoveries are about 60% for both drugs. The coefficient of variation for intra-assay precision, inter-assay precision and accuracy is less than 10% for sulfadoxine (10-100 microg/ml) and pyrimethamine (1-10 microg/ml).     

Kinetic and quantitative measurements of catecholamine transport in chromaffin ghosts using a glassy carbon electrode

A method for the on-line kinetic measurement of net catecholamine uptake and release in ghosts derived from bovine chromaffin granules is described. Changes in free catecholamine concentration in 1 to 2 ml of media containing chromaffin ghosts were continuously measured through the amperometric detection of their oxidation products through a glassy carbon electrode set at 0.5-V potential vs a reference electrode. Parallel measurements of catecholamine uptake and release in the ghosts under various metabolic conditions show a good quantitative agreement between the values obtained with the electrode and those obtained through high-performance liquid chromatography after separation of the ghosts from the medium. Initial velocities of ATP-dependent uptake of epinephrine, norepinephrine, dopamine, and isoproternol by the ghosts are shown. This method permits, for the first time, quantitation of unidirectional movement of catecholamines in the presence of minute quantities of biological samples. The advantages, limitations, and suitability of this method to measure catecholamine transport in other systems are discussed.     

BRAIN TISSUE AND PLASMA ASSAY OF L-DOPA AND α-METHYLDOPA METABOLITES BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

Using reverse phase high-performance liquid chromatography and electrochemical detection with mobile phases composed of simple acids, we have developed an assay technique to measure multiple catecholamines and their catechol metabolites in plasma or brain tissue with sensitivity to the picomole level. Ion-pairing chromatography with nitric or trichloroacetic acid as the mobile phase permits separation and quantitation of norepinephrine, α-methylnorepinephrine, epinephrine, dopamine, α-methyldopamine, l -DOPA, α-methyldopa, carbidopa, and DOPAC. Alumina extraction selectively isolates catechols which are then separated on a reverse-phase column and measured by a commercially available electrochemical detector. This method has been applied to measurement of L-DOPA metabolites in patients with Parkinson's disease treated with L-DOPA and carbidopa and to measurement of catecholamines in rat hypothalamus in the course of studies on L-DOPA and α-methyldopa metabolism. Dihydroxybenzylamine is added as an internal standard and standard curves are linear over two orders of magnitude in concentration with coefficients of variation averaging 3.1%. Quantitation is routinely done to 20 pmol with absolute sensitivity possible to 0.5 pmol.     

Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

Study of salivary catecholamines using fully automated column-switching high-performance liquid chromatography

Cortisol and catecholamines are major physiological markers of human stress. In order to establish a fully automated assay system for both cortisol and catecholamines in saliva, which can be sampled without imposing stress, the previously developed system for salivary cortisol [Okumura et al., J. Chromatogr. B, 670 (1995) 11] was modified. The practical sensitivity was around 0.1 pmol ml⁻¹ for norepinephrine and epinephrine and 0.5 pmol ml⁻¹ for dopamine. The established assay procedure provided R.S.D. values of 2∼3% and recoveries of 96∼104% at 0.5 pmol ml⁻¹. Measurement of salivary catecholamines in more than 300 samples taken from about 50 healthy volunteers indicated that the normal values of norepinephrine and dopamine were very low, about 0.1 pmol ml⁻¹ each. In contrast to cortisol, salivary catecholamine levels did not parallel those in plasma. Nevertheless, since levels of salivary catecholamines may reflect the sympathetic nerve activity in the salivary gland, they were assayed in volunteers making a scientific presentation before a large audience. Four out of eleven volunteers reported strong feelings of fear or anxiety, and their salivary catecholamine levels were about ten times higher than normal.     

Determination of ketamine and norketamine enantiomers in plasma by solid-phase extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method is described for determination of sub-anaesthetic concentrations of the enantiomers of ketamine and its metabolite norketamine in plasma. The samples are purified by reversed-phase solid-phase extraction. The enantiomers are separated on a Chiral AGP column with a mobile phase containing 16% methanol and a 10 mM phosphate buffer at pH 7.0, and measured by UV-detection at a wavelength of 220 nm. Linear calibration curves with correlation coefficients better than 0.995 have been obtained in the range 10–320 ng/ml. Minimum detectable concentrations were about 2 ng/ml.