Pharmacokinetics of 19-nortestosterone esters in normal men

A reliable method for the isolation of 19-nortestosterone (NT), testosterone (T) and dihydrotestosterone (DHT) by high-performance liquid chromatography (HPLC) and quantitation of the individual steroids by radioimmunoassays is described. The method was used to measure serum concentrations of NT, T and DHT in a pharmacokinetic study and in a clinical trial for male fertility control. Following intramuscular injection of either 50 mg 19-nortestosterone-3-(p-hexoxyphenyl)-propionate (NP) or 50 mg 19-nortestosterone-decanoate (ND) serum NT increased rapidly to maximal concentrations of 4.6 +/- 3.2 and 2.0 +/- 1.3 nmol/l (+/-SD), respectively, in the 6 volunteers. The half-life time was 8 days for ND and 21 days for NP. Based on these findings a clinical trial with NP was performed. NP was given to 5 healthy men in doses of 100 mg/week for the first 3 weeks followed by 200 mg/week for 10 further weeks. Serum NT levels increased gradually and maximal concentrations were reached in the 13th treatment week (20.2 +/- 3.4 nmol/l). Measurable amounts of NT were detectable for 19 weeks after the last injection. The study shows that NT accumulates under this treatment regime and wider spacing of the injection intervals may be possible in future trials.     

Tetrazolium salts: a consumer's guide

Synopsis The purities of seven tetrazolium salts, obtained from various commercial sources, have been assessed by thin layer chromatography, relative extinction coefficients, and melting points. MTT and INT were largely homogeneous on thin layer chromatography, although significant variations occurred in the melting point behaviour. All the samples of TT examined were contaminated to a small extent with non-tetrazolium u.v.-absorbing material. TNBT and NBT were contaminated with small amounts of mono-tetrazolium salts, although one sample of each was heavily contaminated with another di-tetrazolium compound. Four samples of TNBT contained high melting point contaminants. BT was also contaminated with mono-tetrazolium salts, and some samples also contained di-tetrazolium salt contaminants. NT was the most heavily contaminated of all, most samples containing no less than five separate tetrazolium compounds. Prices varied widely, and in general were not related to purity. Some catalogue entries were very easy to find; others were more difficult. Few specifications were given; of these, most were arbitrary (for example, pure, grade I, and ... probably the finest INT offered anywhere).     

The measurement of dimethylamine, trimethylamine, and trimethylamine N-oxide using capillary gas chromatography-mass spectrometry

We have developed a method for measuring dimethylamine (DMA), trimethylamine (TMA), and trimethylamine N-oxide (TMAO) in biological samples using gas chromatography with mass spectrometric detection. DMA, TMA, and TMAO were extracted from biological samples into acid after internal standards (labeled with stable isotopes) were added. p-Toluenesulfonyl chloride was used to form the tosylamide derivative of DMA. 2,2,2-Trichloroethyl chloroformate was used to form the carbamate derivative of TMA. TMAO was reduced with titanium(III) chloride to form TMA, which was then analyzed. The derivatives were chromatographed using capillary gas chromatography and were detected and quantitated using electron ionization mass spectrometry (GC/MS). Derivative yield, reproducibility, linearity, and sensitivity of the assay are described. The amounts of DMA, TMA, and TMAO in blood, urine, liver, and kidney from rats and humans, as well as in muscle from fishes, were determined. We also report the use of this method in a pilot study characterizing dimethylamine appearance and disappearance from blood in five human subjects after ingesting [13C]dimethylamine (0.5 mumol/kg body wt). The method we describe was much more reproducible than existing gas chromatographic methods and it had equivalent sensitivity (detected 1 pmol). The derivatized amines were much more stable and less likely to be lost as gases when samples were stored. Because we used GC/MS, it was possible to use stable isotopic labels in studies of methylamine metabolism in humans.     

Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10–15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.     

Properties of halophil nicotinamide–adenine dinucleotide phosphate-specific isocitrate dehydrogenase. True Michaelis constants, reaction mechanisms and molecular weights

True values of Michaelis constants of the NADP(+)-specific isocitrate dehydrogenase from Halobacterium salinarium were not very different from those of the apparent constants reported by Aitken et al. (1970). The true constants were affected by salt in a similar manner to that of the apparent constants obtained with NADP(+) at fixed concentrations of 1.0-0.2mm and threo-d(s)-(+)-isocitrate at fixed concentrations of 2.0-0.125mm. The response of apparent V(max.) to salt concentration was highly dependent on fixed substrate concentration in solutions of sodium chloride but much less so in solutions of potassium chloride. At several levels the results emphasize the difficulty of generalizing about the salt relations of a halophil enzyme without adequate attention to substrate concentration. The enzyme has at least two different reaction mechanisms depending on salt concentration. In its ;physiological' form (i.e. in 1.0m-potassium chloride), and also in 1.0m-sodium chloride, the reaction mechanism is ordered with NADP(+) the first substrate added and NADPH the last product released. In 0.25m-sodium chloride, however, the mechanism is different and is probably non-sequential. In 4.0m-sodium chloride with low concentrations of either fixed substrate, there was evidence of a co-operative action of the variable substrate. The evidence suggests that salt participates in the reaction mechanism in two ways: one is the reversible addition to the enzyme in a manner analogous to that of a substrate; the other is dead-end complex-formation. The relative contributions of these two types of reaction determine whether salt activates or inhibits the enzyme. In addition, the inhibition caused by high concentrations of sodium chloride is more complex than the corresponding inhibition by potassium chloride. Gel-filtration experiments indicated that at very low salt concentrations the enzyme has an apparent molecular weight of about 70800. In ;physiological' concentrations of potassium chloride the enzyme appears to be a dimer (mol.wt. 122000-135000) and, in 1.0-4.0m-sodium chloride, it behaves as a trimer or tetramer (mol.wt. 224000-251000). A preliminary method of purifying the enzyme is described.     

Stereoselective analysis of ketorolac in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) analytical method is described for the quantification of the (R)- and (S)-enantiomers of ketorolac when present together in human plasma. The method involves derivatization with thionyl chloride/(S)-1-phenylethylamine and subsequent reversed-phase chromatography of the diastereomeric (S)-1-phenylethylamides of (R)- and (S)-ketorolac. The method is suitable for the analysis of large numbers of plasma samples and has been applied in this report to a pharmacokinetic study of ketorolac enantiomers upon intramuscular administration of racemic drug to a human subject. The limit of quantification for each enantiomer of ketorolac is 50 ng/ml (signal-to-noise ratio > 10). © 1993 Wiley-Liss, Inc.     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine with thiosulfate: synthesis of L-alanine sulfodisulfane and application to the determination of thiosulfate

A new reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine (ACESC) with thiosulfate is described. The reaction proceeded quantitatively in formic or acetic acid solutions, yielding equimolar amounts of L-alanine sulfodisulfane (2-amino-2-carboxyethyl sulfodisulfane) and L-alanine 3-sulfinic acid. L-Alanine sulfodisulfane was obtained as pure monosodium salt; the yield was 92% of the theoretical. A new method is described for the determination of thiosulfate. The method is based on the quantitative reaction between ACESC and thiosulfate, and L-alanine sulfodisulfane, one of the reaction products, was determined using acid ninhydrin reagent 2 of M. K. Gaitonde (1967, Biochem. J. 104, 627-633). The recovery was over 95%. When samples contained sulfite in addition to thiosulfate, S-sulfo-L-cysteine (T. Ubuka et al., 1982, Anal. Biochem. 126, 273-277) was produced in addition to L-alanine sulfodisulfane by the treatment with ACESC. Both products were separated by a small Dowex 1 column and determined with the acid ninhydrin reagent 2. The recoveries were over 95%. The new method was applied to the thiosulfate sulfurtransferase reaction, in which thiosulfate, a substrate, and sulfite, a product, were determined separately.     

Proneurotensin 1-117, a stable neurotensin precursor fragment identified in human circulation

Proneurotensin/neuromedin N (pro NT/NMN) is the common precursor of two biologically active peptides, neurotensin (NT) and neuromedin N (NMN). We have established antibodies against peptide sequences of the NT/NMN precursor and developed a sandwich immunoassay for the detection of pro NT/NMN immunoreactivity in human circulation. Endogenous pro NT/NMN immunoreactivity was enriched by affinity chromatography using antibodies against two different pro NT/NMN epitopes, and further purified by reversed phase HPLC. Mass spectrometry analysis revealed pro NT/NMN 1-117 as major pro NT/NMN immunoreactivity in human circulation. Pro NT/NMN 1-117 is detectable in serum from healthy individuals (n = 124; median 338.9 pmol/L). As known for NT, the release of pro NT/NMN 1-117 from the intestine into the circulation is stimulated by ingestion of an ordinary meal. Investigation of the pro NT/NMN 1-117 in vitro stability in human serum and plasma revealed that this molecule is stable for at least 48 h at room temperature. Since pro NT/NMN 1-117 is theoretically produced during precursor processing in stoichiometric amounts relative to NT and NMN, it could be a surrogate marker for the release of these bioactive peptides.     

A novel assay for the biosynthesis of sulphated polysaccharide and its application to studies on the effects of somatomedin on cultured cells

1. A simple method is described for the determination of small amounts of [(35)S]sulphated polysaccharide with 95-100% recovery in the range from 0.3 to 150mug of polysaccharide. 2. The method is based on precipitation with cetylpyridinium chloride of polysaccharide samples applied to filter paper. It is not significantly disturbed by the presence in the sample of a large excess of inorganic (35)SO(4) (2-). 3. Sulphated glucosamino- and galactosaminoglycans may be determined separately by treatment of the sample with chondroitinase ABC. 4. The method is applicable to the assay of [(35)S]sulphated polysaccharide biosynthesis in cell cultures. A stimulation of sulphate incorporation obeying a linear dose-response curve, was demonstrated in somatomedin-incubated fibroblast and glia cell cultures. 5. The described system provides a new assay method for somatomedin. 6. The stimulatory effect of somatomedin on the synthesis of [(35)S]sulphated polysaccharide appeared to be general, rather than specific, for a particular type of polysaccharide.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)- -cysteine with thiosulfate: Synthesis of -alanine sulfodisulfane and application to the determination of thiosulfate

A new reaction of S-(2-amino-2-carboxyethylsulfonyl)- -cysteine (ACESC) with thiosulfate is described. The reaction proceeded quantitatively in formic or acetic acid solutions, yielding equimolar amounts of -alanine sulfodisulfane (2-amino-2-carboxyethyl sulfodisulfane) and -alanine 3-sulfinic acid. -Alanine sulfodisulfane was obtained as pure monosodium salt; the yield was 92% of the theoretical. A new method is described for the determination of thiosulfate. The method is based on the quantitative reaction between ACESC and thiosulfate, and -alanine sulfodisulfane, one of the reaction products, was determined using acid ninhydrin reagent 2 of [7.]. The recovery was over 95%. When samples contained sulfite in addition to thiosulfate, S-sulfo- -cysteine ([6.]) was produced in addition to -alanine sulfodisulfane by the treatment with ACESC. Both products were separated by a small Dowex 1 column and determined with the acid ninhydrin reagent 2. The recoveries were over 95%. The new method was applied to the thiosulfate sulfurtransferase reaction, in which thiosulfate, a substrate, and sulfite, a product, were determined separately.     

Plate height determination for gradient elution chromatography of proteins

A method for determining the plate height HETP from the elution curve obtained by the linear gradient elution (LGE) ion-exchange chromatography (IEC) of proteins is presented. The method was developed on the basis of the numerical solutions of a chromatography model which considers the zone sharpening and the distribution coefficient as a function of the salt concentration. The plate height HETP is determined from the peak width and the salt concentration at which the peak is eluted in LGE. The method was applied to the experimental results with various ion-exchange chromatography media. A calculation example based onthe present method is presented to show how the chromatographic and operating parameters should be tuned to obtain a desired resolution. A simplified calculation procedure for the peak profile is also described. (c) 1995 John Wiley & Sons, Inc.     

Changes in urinary water and electrolyte excretion in sodium-loaded sheep in response to intravenous infusion of arginine vasopressin.

Mature sheep receiving supplements of sodium chloride into the rumen were given intravenous infusions of arginine vasopressin at rates varying from 4-6-23 pmol/min (2-10 mU/min). Infusion of the hormone led to an increase in urine flow and to increases in the amounts of sodium and chloride excreted, the effect on flow was, however, the greater so that the osmolality of the urine fell during the infusions. In sheep given intravenous infusions of a hypertonic sodium chloride solution addition of vasopressin to the infusate led to the formation of a larger volume of urine containing a higher proportion of the infused salt load compared to when the salt solution alone was given. As before the effect on flow was the greater and hence the osmolality of the urine was lower when the hormone was given. In other experiments intravenous infusion of a hypertonic sodium chloride solution at rates providing 2-8 mmol NaCl/min led to increases in urine flow and increases in sodium and chloride excretion, the size of these increases being proportional to infusion rate. Plasma vasopressin levels markedly increased during these infusions, the levels seen being similar to those seen in sheep given vasopressin in amounts which increased both urine flow and electrolyte excretion. This suggests that during hypertonic salt loading vasopressin probably contributes directly to the increases in urine flow and the increases in electrolyte excretion which are seen. Further evidence in support of this was obtained in experiments in which a greater natriuretic response was seen in sheep given a hypertonic sodium chloride solution into the carotid artery as opposed to the given a hypertonic sodium chloride solution into the carotid artery as opposed to the jugular vein and where it was shown that plasma vasopressin levels were indeed higher when the solution was given into the artery.     

Characterization of human plasma neurotensin-like immunoreactivity after fat ingestion

The concentration of neurotensin-like immunoreactivity in plasma (p-NTLI) increases after the ingestion of food, and fat seems to be the most important nutrient. It is essential to characterize the NT species that are responsible for this postprandial rise of p-NTLI. After an overnight fast, two male and two female subjects therefore ingested 300 ml of cream (containing 40% (w/w) milk fat). Unextracted plasma samples were subjected to column chromatography and the eluates were analysed using four NT antisera having different specificities. The concentration of chromatographically identified NT(1-13) in peripheral plasma increased significantly from 3 pM in the fasting state to 26 pM 30 min after the ingestion of fat. The concentration of NT(1-8), which is probably a metabolite of NT(1-13), also increased markedly. No significant increase of smaller COOH-terminal sequences of NT was found. The results show that the plasma concentration of NT(1-13) may increase about tenfold following the ingestion of fat. This is further support for the hypothesis that NT(1-13) may function as a hormone.     

Determination of serum uric acid using high-performance liquid chromatography (HPLC)/isotope dilution mass spectrometry (ID-MS) as a candidate reference method

Uric acid is an important diagnostic marker of catabolism of the purine nucleosides, and accurate measurements of serum uric acid are necessary for proper diagnosis of gout or renal disease appearance. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) has been described. An isotopically labeled internal standard, [1,3-(15)N(2)] uric acid, was added to serum, followed by equilibration and protein removal clean up to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) analyses. (M-H)(-) ions at m/z 167 and 169 for uric acid and its labeled internal standard were monitored for LC/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for uric acid (Standard Reference Materials SRM909b) with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added uric acid. The method performed well against the established reference method of ion-exchange followed by derivatization isotope dilution (ID) gas chromatography mass spectrometry (ID-GC/MS). The results of this method for uric acid agreed well with the certified values and were within 0.10%. The amounts of uric acid recovered and added were in good agreement for the three concentrations. This method was applied to determine uric acid in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.08-0.18% and between-set CVs of 0.02-0.07% for LC/MS analyses. Liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analysis was also performed. The LC/MS and LC/MS/MS results were in very good agreement (within 0.14%). This LC/MS method, which demonstrates good accuracy and precision, and is in the speed of analysis without the need for a derivatization stage, qualifies as a candidate reference method. This method can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Peroxynitrite diminishes myogenic tone in cerebral arteries: role of nitrotyrosine and F-actin

This study investigated the effect of peroxynitrite (OONO(-))-induced nitrosylation of filamentous (F)-actin on myogenic tone in isolated and pressurized posterior cerebral arteries (PCAs). Immunohistochemical staining was used to determine 3-nitrotyrosine (NT) and F-actin content in vascular smooth muscle after exposure to 10(-7) M or 10(-4) M OONO(-) for 5 or 60 min in isolated third-order PCAs (n = 37) from male Wistar rats pressurized to 75 mmHg in an arteriograph chamber, quantified with confocal microscopy. Additionally, the role of K(+) channels in OONO(-)-induced dilation was investigated with 3 microM glibenclamide or 10 mM tetraethylammonium chloride before OONO(-) exposure. OONO(-) (10(-4) M) induced a 40% dilation of tone (P < 0.05) while diminishing F-actin content by half (P < 0.05) and causing a 60-fold increase in NT (P < 0.05) in the vascular smooth muscle of PCAs. Additionally, F-actin was inversely correlated with both diameter and NT content (P < 0.05) and was significantly colocalized in the vascular smooth muscle with NT (overlap coefficient = 0.8). The dilation to ONOO(-) was independent of K(+) channel activity and thiol oxidation as glibenclamide, tetraethylammonium chloride, and dithiothreitol had no effect on OONO(-)-induced dilation or F-actin or NT content in PCAs. Because NT was colocalized with F-actin, we hypothesize that OONO(-) induces nitrosylation of F-actin in vascular smooth muscle leading to depolymerization and the subsequent loss of myogenic tone, which may promote vascular damage during oxidative stress such as in ischemia and reperfusion injury.     

A rapid and sensitive method for the analysis of carbohydrate components in glycoproteins using gas-liquid chromatography

A rapid and simple gas-liquid chromatographic method for the determination of subnanomolar amounts of carbohydrates derived from glycoproteins is described. The procedure involves methanolysis in the presence of methyl acetate followed by removal of hydrogen chloride by coevaporation with t-butyl alcohol and trimethylsilylation. The method is also applicable to samples containing uronic acids and lipids.     

Gas chromatographic determination of inorganic tin in rat urine after a single oral administration of stannous chloride and mono-, di-, and triphenyltin chloride

A method is described for the determination of inorganic tin by gas chromatography with flame photometric detection. The inorganic tins, stannous and stannic, were extracted with hydrochloric acid and n-hexane—benzene in the presence of 0.05% tropolone, and both inorganic tins were pentylated to tetrapentyltin with a Grignard reagent prior to gas chromatography. The absolute limit of detection for tetrapentyltin was 3 pg as tin. The recovery of stannous chloride added to rat urine samples was 80.2 ± 2.4% (mean ± S.D., n = 8). The application of this method to the study of urinary excretion of inorganic tin and organotin compounds in rats following oral administration of tin compounds is presented. The urinary excretion of tin compounds was observed over a period of 96 h following administration of stannous chloride or phenyltin compounds. Most of the inorganic tin was excreted into urine within 24 h after administration of stannous chloride. In the experiments on organotin administration, the level of the excretion as total tin for monophenyltin reached a maximum ca. 0–24 h after administration, whereas the maxima for di- and triphenyltin were found after 24–48 h and 48–72 h, respectively. The predominant excretion product of these tin compounds found in urine was monophenyltin.     

Trizol-based method for sample preparation and isoelectric focusing of halophilic proteins

A persisting complication in the development of well-resolved two-dimensional PAGE maps of halophilic proteins is their natural incompatibility with isoelectric focusing (IEF). The complete desalting of samples, which is necessary for IEF, tends to aggregate halophilic proteins, often requires relatively large amounts of starting material due to significant loss of sample, and is relatively time-consuming. Here, we describe a method of preparing protein samples from the haloarchaeon Haloferax volcanii that not only desalts the samples thoroughly but also drastically reduces the amount of protein loss associated with previous sample preparation methods and prevents protein aggregation during the removal of salt. This method of sample preparation, which incorporates Trizol (phenol/guanidine isothiocyanate), can easily be extended to analyze halophilic proteins from other organisms.     

Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.

A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.