Urinary excretion of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA), including the sulfate and glucuronide of 4-hydroxy-3-methoxymethamphetamine (HMMA), in humans and rats.

The urinary concentrations of the main metabolites of 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy), specifically 4-hydroxy-3-methoxymethamphetamine sulfate (HMMA-Sul) and 4-hydroxy-3-methoxymethamphetamine glucuronide (HMMA-Glu), have been directly measured in both MDMA users and rats by an established liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) procedure. The concentrations of these conjugates in urine from MDMA users (n = 25) ranged from 6.5 to 202 microM (from 1.8 to 55.6 microg ml(-1)) for HMMA-Sul and from 1.3 to 87.0 microM (from 0.5 to 32.3 microg ml(-1)) for HMMA-Glu, and the ratio of HMMA-Sul to HMMA-Glu ranged from 1.6 to 9.9 (3.1 +/- 1.8). These results demonstrate that the sulfation is quantitatively more significant than the glucuronidation for HMMA in humans. In rats, in contrast, almost all the conjugated HMMA (>99%) was excreted as the glucuronide. These findings indicate that hydrolysis should be carefully made in urine analysis by gas chromatography (GC) or gas chromatography-mass spectrometry (GC-MS) by using either an acid or an enzyme possessing both sulfatase and beta-glucuronidase activities. It is concluded that a considerable interspecies variation exists in the conjugation of HMMA between humans and rats.

Alterations in D-amino acid levels in the brains of mice and rats after the administration of D-amino acids.

To mutant ddY/DAO(-) mice lacking D-amino-acid oxidase activity and normal ddY/DAO(+) mice, five D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu and D-Pro) were orally administered for two weeks, and the D-amino acid levels were examined in seven brain regions. The levels of D-Asp markedly increased in the pituitary and pineal glands in both strains. In the ddY/DAO(+) mice, the levels of the other D-amino acids did not significantly change in most of the brain regions. While in the ddY/DAO(-) mice the levels of D-Ser significantly increased in most of the brain regions except for the cerebrum and hippocampus. The levels of D-Ala and D-Leu increased in all regions but the levels of D-Pro did not significantly change. The same five D-amino acids were intravenously injected into Wistar rats and the D-amino acid levels in their brains were examined for 60 min after the administration. The levels of D-Asp markedly increased in the pineal gland 3 min after the administration, while the levels of D-Ser, D-Ala, and D-Pro increased both in the pineal and pituitary glands, the levels of D-Leu increased in all brain regions. These results are useful for the elucidation of the origins and regulation of D-amino acids in the mammalian body.

Metabolism of the recently encountered designer drug, methylone, in humans and rats.

The urinary metabolites of methylone in humans and rats were investigated by analysing urine specimens from its abuser and after administrating to rats with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-electrospray ionization mass spectrometry (LC-ESI MS), using authentic standards. The time-course excretion profiles of methylone and its three metabolites in rats were further investigated after a single intraperitoneal dosing of 5 mg kg-1 methylone hydrochloride. Two major metabolic pathways were revealed for both humans and rats as follows: (1) side-chain degradation by N-demethylation to the corresponding primary amine methylenedioxycathinone (MDC), partly conjugated; and (2) demethylenation followed by O-methylation of either a 3- or 4-OH group on the benzene ring to produce 4-hydroxy-3-methoxymethcathinone (HMMC) or 3-hydroxy-4-methoxymethcathinone (3-OH-4-MeO-MC), respectively, mostly conjugated. Of these metabolites, HMMC was the most abundant in humans and rats. The cumulative amount of urinary HMMC excreted within the first 48 h in rats was approximately 26% of the dose, and the amount of the parent methylone was not more than 3%. These results demonstrate that the analysis of HMMC will be indispensable for proof of the use of methylone in forensic urinalysis.

Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice.

Determination of free D-aspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino acid oxidase activity.

A simple and precise method for the simultaneous determination of free D-aspartic acid, D-serine and D-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-L-cysteine. With the present method, the contents of these three D-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking D-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of D-serine were observed in forebrain (100-400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, D-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, D-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.

A sensitive assay of human blood platelet cyclic nucleotide phosphodiesterase activity by HPLC using fluorescence derivatization and its application to assessment of cyclic nucleotide phosphodiesterase inhibitors.

A selective and sensitive HPLC measurement of 3',5'-cyclic nucleotide phosphodiesterase (PDE) activity in human platelets using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorogenic reagent for guanine and its nucleosides and nucleotides is described. cGMP, a substrate for PDE, and GMP, which was produced by the enzyme reaction, are selectively converted by the reaction with DMPG to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. Human platelet PDE activity was measured and the inhibitory effects of several compounds were investigated.

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization.

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.

Determination of minute amounts of D-leucine in various brain regions of rat and mouse using column-switching high-performance liquid chromatography.

A highly sensitive method for the determination of minute amounts of D-Leu in biological samples was developed. For accurate and sensitive determination, a column-switching system using a micro ODS column and a chiral column was adopted. After pre-column derivatization of D- and L-Leu with NBD-F, the derivatives of the enantiomers were purified on a micro ODS column as a DL mixture. The eluted DL-Leu was then introduced to the chiral column, and each enantiomer was determined. The calibration curve for D-Leu, which was constructed by adding known amounts of D-Leu to a rat hippocampus, was linear from 1 to 1000 fmol (r>0.999), and the detection limit of added D-Leu was 1 fmol (S/N=5). Within-day and day-to-day precisions of D-Leu determination using the same homogenate of rat hippocampus were 5.11 and 5.25% (RSD), respectively. The content of D-Leu in rat hippocampus was 0.69 nmol/g wet tissue (the percentage of D-enantiomer for total Leu was 0.97%), which was consistent with the reported value. The distribution of D-Leu in mouse brain was also investigated, and the presence of D-Leu in various regions of the mammalian brain was first observed.

Chemiluminescence derivatization of methylglyoxal using 2-aminonicotinic acid.

To develop a sensitive and selective chemiluminometric method for the determination of methylglyoxal, we used 2-aminonicotinic acid as the chemiluminescence derivatization reagent. 2-Aminonicotinic acid reacts with methylglyoxal in an acidic solution at 37 degrees C for 4 h and gave a chemiluminescence in N,N'-dimethylformamide containing sodium tert-butoxide. The detection limit (blank intensity plus three-times its standard deviation of methylglyoxal) for methylglyoxal is 233 fmol in the reaction mixture.

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice.

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.

Chemiluminescence characteristics of N-(4-substituted benzyl)isoluminol.

We synthesized N-(4-substituted benzyl)isoluminol which has 4-bromo-, 4-methyl-, 4-methoxy-, 4-nitrogroups. These compounds produced chemiluminescence by the reaction with the oxidizing agent, potassium hexacyanoferrate and hydrogen peroxide, in an alkaline medium. The chemiluminescence intensities of these compounds were 0.03-4.7 times that of isoluminol. We used Hamett substituent constants as a parameter for the electronic substituent effects. The relationship between the amino-H chemical shift value and the Hamett substituent constants showed a good linear correlation. The relationship between the chemiluminescence intensities and the Hamett substituent constants showed a good linear correlation. The relationship between the fluorescence intensities and the Hamett substituent constants also showed a good linear correlation. These results suggest that the change in the electron density around the amino group strongly influences the fluorescence intensities and corresponding chemiluminescence intensities of these derivatives.

Aminopyrazine analogues as chemiluminescence derivatization reagents for pyruvic acid.

Aminopyrazine analogues were studied as sensitive and selective chemiluminescence derivatization reagents for pyruvic acid. These analogues reacted with pyruvic acid under acidic conditions at 100 degrees C to produce Cypridina luciferin derivatives, which exhibit chemiluminescence by reaction with hydrogen peroxide in the presence of potassium t-butoxide in dimethylformamide. Of the four aminopyrazine analogues (2-amino-5-phenylpyrazine, 2-amino-5-(4-hydroxyphenyl)pyrazine, 2-amino-5-(3,4, 5-trimethoxyphenyl)pyrazine, and 2-aminoquinoxaline), in the present test 2-amino-5-(3,4,5-trimethoxyphenyl) pyrazine was the most sensitive for pyruvic acid, and the chemiluminescence intensity was about four times higher than that obtained with aminopyrazine.

Simple and rapid analytical method for carbapenems using capillary zone electrophoresis.

A simple and rapid analytical method for carbapenems using high-performance capillary electrophoresis is described. All therapeutic carbapenem injections in Japan (imipenem, panipenem and meropenem) and four other beta-lactams (piperacillin, cefotiam, cefotaxime, latamoxef) were separated and determined with good repeatability in about 10 min using simple free zone capillary electrophoresis. The electrophoresis buffer was 100 mM phosphate buffer of pH 8.0, and a fused-silica capillary of 25 microm I.D. and 47 cm length was adopted. The present method was successfully applied to monitor the degradation of carbapenems under various conditions (at various temperatures or in coexistence with other drugs prescribed in the case of methicillin-resistant Staphylococcus aureus).

Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography.

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide. The resultant fluorophore was excited at 247 nm and the emission wavelength was 384 nm. The Stokes shift was 137 nm, which was longer than that of melatonin itself (lambda ex 280 nm, lambda em 330 nm). The melatonin derivative was separated by reversed-phase HPLC in about 15 min and the calibration curve was linear from 500 amol to 5 pmol (r > 0.999) with the detection limit of 500 amol (S/N = 5). The sensitivity of this method was about ten times higher than that of previous methods. Both the day-to-day precision and within-day precision were about 5%, and the derivative of melatonin in the aqueous solution was stable for more than 10 days. This method was successfully applied to the determination of melatonin in rat pineal gland.

Coexistence of chronic myelogenous leukemia and multiple myeloma. Case report and review of the literature.

A case report of simultaneous presentation of chronic myelogenous leukemia (CML) and multiple myeloma (MM) in a 72-year-old female is described. Our case was typical of Ph1-positive and chimeric bcr-abl messenger RNA-positive CML. Furthermore, a marked IgG (kappa-type) paraproteinemia was present. Fluorescence in situ hybridization showed that 97% of marrow nucleated cells were positive for bcr-abl fusion signal, when myeloma cells in the bone marrow amounted to 19.0%. In the literature survey, 4 similar cases with coexistence of CML and MM have been identified.

1,2-Diarylethylenediamines as sensitive pre-column derivatizing reagents for chemiluminescence detection of catecholamines in HPLC.

Chemiluminescence detection in high-performance liquid chromatography for derivatives of catecholamines (norepinephrine, epinephrine and dopamine) and isoproterenol was studied on the basis of the peroxyoxalate chemiluminescence reaction. The amines and isoproterenol, derivatized with 1,2-diarylethylenediamines, were separated on a reversed-phase HPLC column (TSK gel ODS-120T) with isocratic elution using a mixture of imidazole buffer (pH 5.8, 120 mM)-methanol-acetonitrile (6:2:9, v/v/v). The eluate was detected by a post-column chemiluminescence reaction system, using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate and hydrogen peroxide. Of the 141,2-diarylethylenediamines investigated, it was found that 1,2-bis(3-chlorophenyl)ethylenediamine, 1,2-bis(3,4-dichlorophenyl)-ethylenediamine and 1,2-bis(4-chlorophyenyl)ethylenediamine were the most sensitive derivatives for all catecholamines. The derivatization and peroxyoxalate chemiluminescence reaction conditions were optimized for 1,2-bis(3-chlorophenyl)-ethylenediamine. The chromatographic detection limits for catecholamines were approximately 40-120 amol for an injection volume of 100 microliters (signal-to-noise ratio of 3).

A practical reagent for reversible amino-protection of insulin, 3,4,5,6-tetrahydrophthalic anhydride.

Of the examined dicarboxylic acid anhydrides, 3,4,5,6-tetrahydrophthalic anhydride was found to be the best reagent in practical use for reversible amino-protection of insulin in terms of the rapidity of acid-deprotection. Twelve Gly(A1),Phe(B1),Lys(B29)-triacyl-insulins were prepared by reaction of porcine insulin with the dicarboxylic acid anhydrides and time courses for the deprotection of the acylated insulins with dilute acetic acid were investigated by means of capillary zone electrophoresis, and the tetrahydrophthalyl(THP)-insulin obtained with the reagent was the most rapidly deacylated (6 h, 25 degrees C, 0.1 M acetic acid). Isolation of triacyl-insulins and Gly(A1)-THP-, Gly(A1),Phe(B1)-diTHP- and Gly(A1),Lys(B29)-diTHP-insulins using DEAE anion-exchange high-performance liquid chromatography is also described.

Preparative and analytical separations by high-performance liquid chromatography and capillary electrophoresis of the products from the reaction of insulin with exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic anhydride.

An anion-exchange high-performance liquid chromatographic method using a TSKgel DEAE-2SW column and zone electrophoresis using a fused-silica capillary are described for the preparative and analytical separations of the products of the reaction between insulin and exo-3,6- epoxy-1,2,3,6-tetrahydrophthalic anhydride (ETPA), an amino protective reagent. Four stable products were identified having exo-3,6-epoxy-1,2,3,6-tetrahydrophthalic group(s) on insulin at the i) Gly(A1), ii) Gly(A1) and Phe(B1), iii) Gly(A1) and Lys(B29), and iv) Gly(A1), Phe(B1) and Lys(B29) sites, together with four labile products produced by the reaction of ETPA with the non-amino groups of the stable products. The usefulness of capillary electrophoresis was demonstrated in the evaluation of the effect of the molar ratios of anhydride to insulin on the production of the insulin derivatives.

Effect of spacer groups connecting insulin with fluorophore in Gly(A1)-sulfobenzoxadiazole-labeled insulins on the immunoreactivity toward anti-insulin antibody.

The immunoreactivity (specific binding) of three Gly(A1)-sulfobenzoxadiazole-labeled insulins which have different spacer groups, Gly(A1)-[3-(4-sulfobenzoxadiazol-7-ylthio)propanoyl]-insulin , Gly(A1)-[2-(4-sulfobenzoxadiazol-7-ylthio)acetyl]-insulin, Gly(A1)-[3-carboxy-2(or 3)-(4-sulfobenzoxadiazol-7-ylthio)propanoyl]-insulin, toward an antiporcine insulin monoclonal antibody was investigated, where solid-phase fluoroimmunoassay technique was utilized. The immunoreactivities of three labeled insulins were 2.1, 1.8 and 1 in that order.

Preparation of fluorescence labeled insulins, sulfobenzoxadiazolyl-insulins, for fluorescence immunoassay.

The preparation of insulins labeled with a fluorescent moiety at a definite position (Gly(A1) or Lys(B29)) on the molecule is described. Gly(A1)- and Lys(B29)-S-acetylthioglycoloyl-insulins and Gly(A1)-2(or 3)-acetylmercapto-3-carboxypropranoyl-insulin were deacetylated and then reacted with 7-chloro-4-sulfobenzoxadiazole, a fluorogenic reagent, to afford the corresponding fluorescence labeled insulins. The preparative separation of the fluorescence labeled insulins from the insulin derivatives that remained unreacted was carried out by anion-exchange high-performance liquid chromatography on a TSKgel DEAE-2SW column.