A novel, quantitative assay for homocarnosine in cerebrospinal fluid using stable-isotope dilution liquid chromatography-tandem mass spectrometry

We describe a rapid and sensitive method for the quantification of homocarnosine in physiological fluids, with particular emphasis on cerebrospinal fluid (CSF). Homocarnosine was quantified as the butyl derivative, with (2)H(2)-l-homocarnosine as internal standard. Following deproteinization of CSF samples, supernatants were evaporated to dryness and derivatized with 10% 6M HCl in butanol. Samples were chromatographed on a C(18) column and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in the multiple reaction monitoring mode. The intra- and inter-assay variations were 4.6 and 10.9%, respectively. Mean recovery of homocarnosine at two concentrations was 105%. The limit of detection in CSF approximated 20 nmol/L. CSF homocarnosine is age dependent and ranges from <0.02 to 10 micromol/L. Our method is applicable to the analysis of CSF derived from patients with heritable defects in the GABA pathway, patients with homocarnosinosis or serum carnosinase deficiency, and should be applicable to other model systems in order to further explore the biological role and significance of homocarnosine in mammalian systems.     

Hydrolysis of carnosine and related compounds by mammalian carnosinases

Comparative study of hydrolysis of carnosine and a number of its natural derivatives by human serum and rat kidney carnosinase was carried out. The rate of carnosine hydrolysis was 3–4-fold higher then for anserine and ophidine. The rate of homocarnosine, N-acetylcarnosine and carcinine hydrolysis was negligible by either of the enzymes used. Our data show that methylation, decarboxylation or acetylation of carnosine increases resistance of the molecule toward enzymatic hydrolysis. Thus, metabolic modification of carnosine may increase its half-life in the tissues.     

A sensitive specific enzymatic-fluorometric assay for homocarnosine

We have developed a sensitive and specific assay for homocarnosine in tissues. Homocarnosine is separated from GABA by ion exchange chromatography. After hydrolysis of homocarnosine with swine kidney carnosinase, the evolved GABA is measured by an enzymatic-fluorometric procedure. As little as 0.1 nmol of tissue homocarnosine can be detected by this procedure. Homoanserine, which would be detected by this assay, can be separated from homocarnosine by thin layer chromatography. No homoanserine could be detected in any tissues examined. There are marked regional variations in levels of homocarnosine in guinea-pig brain that do not correspond to regional differences in GABA levels.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene—divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.     

Carnosine and related compounds protect the double-chain DNA from oxidative damages

Carnosine and its derivatives in the concentrations corresponding to their level in excitable tissues have been shown to protect DNA from oxidative damages. Their efficiency (5 mM) was of the following order: ophidine > carnosine ≈ anserine > homocarnosine > N-acetylcarnosine. β-Alanine and gamma-aminobutyric acid (GABA) did not have any capability for protection. The revealed effect can be one of the causes of oxidative stability of the brain and muscle tissue in vertebrate animals.     

Transport and distribution of homocarnosine after intracerebroventricular and intravenous injection in the rat

Rats were injected intracerebroventricularly (i.c.v.) or i.v. with [¹⁴C]homocarnosine (250 nmol). Distribution of the dipeptide in brain structures, transport from the brain to the blood, distribution in peripheral organs, and excretion in the urine were studied by measuring radioactivity in tissue, plasma, and urine samples by liquid scintillation counting 15–120 min after injection. After i.c.v. injection, [¹⁴C]homocarnosine was taken up into all parts of the brain investigated (highest uptake in structures close to the site of injection), it was transported to the blood, and radioactive substances were found in low concentration in muscle, spleen, and liver, in high concentration in the kidneys, and very high concentration in the urine. Investigations using high pressure liquid chromatography (HPLC) showed that no degradation took place in the brain, all radioactivity was found in the homocarnosine fraction. In the plasma 86% of the radioactivity was found in the GABA fraction presumed to be formed by cleavage of the peptide, while in the kidneys 35% and in the urine 40% was found in the GABA fraction. After i.v. injection of [¹⁴C]homocarnosine, no radioactivity was measured in hippocampus, striatum, cerebellum and cerebral cortex 15 min after injection, however, 60 min after injection a very low activity was detected in these structures (estimated intravascular radioactivity subtracted). A low activity was also measured in the spinal cord both 15 and 60 min after injection. When homocarnosine and GABA were separated on HPLC, all radioactivity in brain tissue was found in the GABA fraction, indicating either that [¹⁴C]homocarnosine did not cross the blood-brain barrier in amounts that could be measured with the method used, or that peptide entering the brain was rapidly transported back to the blood. [¹⁴C]Homocarnosine was not taken up either into crude synaptosomal preparations from hippocampus, striatum, cerebellum, cortex and spinal cord, or into slices prepared from the hippocampus and striatum. Transport from the brain to the kidneys and excretion in the urine seems to be a major route for disposal of this peptide in the rat.     

Chromatographic analysis of the enantiomers of ifosfamide and some of its metabolites in plasma and urine

The enantiomers of the cytostatic drug ifosfamide and the two metabolites 2- and 3-dechloroethylifosfamide were isolated from plasma and urine by liquid-liquid extraction with ethyl acetate, resolved on a Chirasil- -val gas chromatographic column and detected by a nitrogen-phosphorus-selective flame ionisation detector. Resolution of the racemic compounds for identification purposes was also accomplished with high-performance liquid chromatography on a chiral column. The validated gas chromatographic method was suitable to determine the total concentrations and the enantiomeric composition of ifosfamide and its dechloroethylated metabolites in plasma and urine samples from treated patients. Some metabolic preferences in the metabolism of ifosfamide were found.     

Cerebrospinal fluid homocarnosine in Huntington's disease

The concentration of homocarnosine (γ-aminobutyryl-L-histidine) in the cerebrospinal fluid (CSF) of ten patients with Huntington's disease (HD) and 24 control subjects was determined by high-performance cation exchange chromatography. The mean CSF homocarnosine level was significantly lower in HD patients (0.86 ± 0.16 nmo1/m1) than in controls (1.69 ± 0.18 nmo1/m1).     

Analysis of urinary aldosterone metabolites in the rabbit by gas chromatography-mass spectrometry

After a large amount of aldosterone was injected into a male rabbit, urine was collected for 48 h. Separation of urinary aldosterone metabolites into monoglucosiduronate fraction and monosulphate fraction was carried out by a combination of countercurrent distribution and DEAE-Sephadex A-25 column chromatography. Each fraction was hydrolyzed with enzyme and free steroids released were separated by Sephadex LH-20 column chromatography. The free steroid was then identified by gas chromatography-mass spectrometry. In monoglucosiduronate fraction, 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were found. On the other hand, 3 alpha, 5 beta-tetrahydroaldosterone was the only aglycone detected in monosulphate fraction. These findings comfirmed results in the preceding paper, where the free steroid was characterized on the basis of the mobility of the steroid and its derivatives on paper chromatography.     

Identification of (23S)-5 alpha-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol in urine of patients with cerebrotendinous xanthomatosis.

This paper describes the identification of a new bile alcohol possessing the 5 alpha-cholestane structure that was found in the urine of patients with cerebrotendinous xanthomatosis. The urine samples were extracted with reversed-phase resin, treated with beta-glucuronidase, and separated on silica gel and reversed-phase column chromatography. The new bile alcohol isolated was the second component of the urinary bile alcohols and was identified as (23S)-5 alpha-cholestane-3 alpha,7 alpha,12 alpha,23,25-pentol by means of gas-liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopic studies.     

Purification of a young age-related glycoprotein (Ugl-Y) from normal human urine

Ugl-Y is a glycoprotein that is detected in normal urine samples from young men and women aged 0 to 17 years. It was purified by ammonium sulfate precipitation and various column chromatographies including affinity chromatography using anti-adult urine antibody coupled to Sepharose 4B. The homogeneity of the glycoprotein was confirmed by polyacrylamide gel electrophoresis, isoelectric focusing, column chromatography on Sephadex G-75, and the precipitation reaction with anti-Ugl-Y antibody. It was shown to have a molecular weight of 29,000 by gel filtration, and to contain 5.2% neutral sugars (mannose and galactose) and 4% hexosamine (glucosamine). Amino acid analysis of the glycoprotein indicated high contents of acidic and hydroxylic amino acids. Its origin is unknown.     

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.     

New metabolites of riboflavin appear in human urine

By surveying compounds having isoalloxazine derived from flavins on a high performance liquid chromatogram with fluorescence detection, two new flavin derivatives were found in human urine. These two compounds were purified by partition chromatography on a cellulose column and by paper chromatography with several solvent systems, and their structures were determined to be 7 alpha-hydroxyriboflavin and 8 alpha-hydroxyriboflavin. The relative distributions, measured by high performance liquid chromatography, of 7 alpha- and 8 alpha-hydroxyriboflavin, riboflavin, and hydroxyethylflavin and its derivative were calculated to be 31.1, 5.0, 25.6, 4.9, and 21.9%, respectively, to total flavins in normal human urine obtained in early morning. The excretion of 7 alpha- and 8 alpha-hydroxyriboflavin in human urine indicates the occurrence of a metabolic pathway of the isoalloxazine ring of flavin at its 7 alpha and 8 alpha positions.     

A urinary pentasaccharide in bovine mannosidosis.

Abnormally high amounts of low molecular weight mannose-rich carbohydrate material were found in the urine of an Angus calf with mannosidosis. At least five oligosaccharide fractions were detected by paper chromatography. The most abundant compound was purified by gel chromatography, zone electrophoresis, and two consecutive preparative paper chromatographic steps. The yield was 10 mg/liter of urine. From structural studies including nuclear magnetic resonance spectroscopy, optical rotation, sugar analysis, methylation analysis, and partial enzymatic degradation the following structure was deduced: alpha-D-Manp-(1 leads to 6)-beta-D-Manp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to 4)-D-GlcNAc. This oligosaccharide is distinct from all the oligosaccharides previously described which are excreted by patients with mannosidosis.     

Metabolic Transformation of Neuropeptide Carnosine Modifies Its Biological Activity

1. The ability of carnosine and carnosine-related compounds (CRCs) to interact with several free oxygen radicals is analyzed.2. Carnosine, the CRCs (imidazole, histidine, anserine), and ergothioneine were found to be equally efficient in singlet oxygen quenching. During generation of hydroxyl radicals from hydrogen peroxide in the Fenton reaction, carnosine was found to be more effective than the CRCs tested.3. By measuring the chemiluminescence produced by carnosine and CRCs in rabbit leukocytes in the presence of luminol or lucigenin, we conclude that carnosine and other CRCs play a stimulating role in superoxide oxygen production while suppressing the myeloperoxidase system.4. ADP-induced aggregation of human platelets is slightly stimulated by carnosine but is inhibited by acetylanserine.5. The following rank order of efficiency of CRCs was demonstrated while measuring the oxidation of human serum lipoproteins: acetylcarnosine < acetylanserine < homocarnosine = ophidine < carnosine < anserine.6. The results obtained demonstrate that metabolic transformation of carnosine into CRCs in tissues may play an important role in regulating the native antioxidant status of the organism.     

High-performance liquid chromatographic separation and isolation of quinidine and quinine metabolites in rat urine

A procedure for the separation and isolation of the urinary metabolites of quinidine and quinine by reversed-phase high-performance liquid chromatography is described. Nine metabolites of quinidine and eight metabolites of quinine were detected in the urine of male Sprague-Dawley rats after a single dose of quinidine or quinine (50 mg kg^?1). Following extraction from urine, the metabolites were separated on either an analytical or a semi-preparative reversed-phase column by gradient elution. After isolation and derivatization, the metabolites were analyzed by gas chromatography and gas chromatography—mass spectrometry.     

Measurement of epsilon-N-trimethyllysine in human blood plasma and urine

A method for measurement of epsilon-N-trimethyllysine in human blood plasma and urine is described. An internal standard, delta-N-trimethylornithine, was added to plasma and urine specimens and the mixtures were deproteinized and/or hydrolyzed. Preliminary purification of epsilon-N-trimethyllysine and delta-N-trimethylornithine was achieved by sequential cation-exchange--anion-exchange chromatography. Amino acids in the column eluates were derivatized with o-phthalaldehyde and mercaptoethanol, and were separated by isocratic reversed-phase high-performance liquid chromatography in the presence of an ion-pairing reagent. Quantitation was achieved by post-column fluorometry. The limit of detection was 5 pmol of epsilon-N-trimethyllysine injected into the chromatograph. The procedure was suitable for determination of epsilon-N-trimethyllysine in 1 ml of plasma or 0.2-0.4 ml of urine. The method was applied to measurements of epsilon-N-trimethyllysine in plasma and urine of four systemic carnitine deficiency patients and six normal subjects. Plasma epsilon-N-trimethyllysine concentration was significantly lower in systemic carnitine deficiency patients compared to normal individuals, but no significant difference in urinary epsilon-N-trimethyllysine excretion was observed between the two groups.     

Sample clean-up with sol-gel enzyme and immunoaffinity columns for the determination of bisphenol A in human urine

The paper describes the development of a simple and highly selective analytical method for the determination of free and total bisphenol A in urine samples. Free bisphenol A levels can be determined after sample clean-up using sol-gel immunoaffinity columns containing anti-bisphenol A antibodies. In determining total bisphenol A levels, the sample pre-treatment procedure consists of sample preparation using an on-line combination of two sol-gel columns, an enzyme column containing glucuronidase and arylsulfatase, and an immunoaffinity column. Bisphenol A can then be quantified by high-performance liquid chromatography and fluorescence detection. The mean recovery was found to be 78% with a standard deviation of 3.4%, the LOD (S/N=3) was 0.2 ng/ml. The method was applied to determine free and total urinary BPA levels of healthy adults and dialysis patients.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

Isolation and identification of some guanidino compounds in the urine of patients with hyperargininaemia by liquid chromatography,thin-layer chromatography and gas chromatography-mass spectrometry

Liquid column chromatographic studies of monosubstituted guanidino compounds, which are excreted in the urine of patients with hyperargininaemia are reported. The guanidino-positive peaks, with the highest excretion values, were isolated from urine and the isolated compounds were identified by thin-layer chromatography and gas chromatography—mass spectrometry. Guanidinoacetic acid, N-α-acetylarginine, argininic acid, γ-guanidinobutyric acid, arginine and α-keto-δ-guanidinovaleric acid were found to be excreted at high levels in the urine of patients with hyperargininaemia compared with controls.