Identification of fendiline metabolites in human urine

After oral application of 14C labelled fendiline, 13 metabolites of this drug could be identified in human urine. Only traces of parent fendiline were excreted in the urine. The main pathway of metabolism is hydroxylation of phenyl groups with subsequent glucuronidation and sulphation. On the other hand, oxidative dealkylation occurs with the amino group remaining at the 3,3-diphenylpropyl moiety and p-hydroxyacetophenone being formed almost entirely from the 1-phenylethyl group.     

Identification of 2-guanidinoethanol in human urine

Guanidino compounds in normal human urine were analyzed by high-performance liquid chromatography; an unknown peak was observed in the chromatogram that was identical to the peak of synthetic 2-guanidinoethanol. In another experiment, the substance was purified from human urine by successive use of strongly acidic ion-exchanger, thin-layer chromatography and then weakly acidic ion-exchanger. After this it was reacted with acetylacetone to form dimethylpyrimidyl derivative. After further reaction of this derivative with trifluoroacetic anhydrate, it was analyzed by gas chromatography/mass spectrometry. The mass chromatogram and mass spectrum were identical to those of the trifluoroacetylated dimethylpyrimidyl derivative of synthetic 2-guanidinoethanol. This is the first report on the identification of 2-guanidinoethanol in human urine. The concentration of 2-guanidinoethanol in the urine of healthy humans was 5.7 +/- 1.8 (mean +/- SD) mumol/g creatinine.     

Characterization of a new flavin metabolite from human urine

A new flavin metabolite comprising approximately 5% of the total flavin of human urine was isolated and characterized using absorption and fluorescence spectra, oxidation-reduction and hydrolysis data, and ninhydrin reactions. The flavin is a derivative associated with a peptide residue in ester linkage from an amino acid carboxyl to the ribityl chain of riboflavin, probably at the 5'-terminus.     

Methionyl-lysyl-bradykinin, a new kinin from ox blood

1. The pseudoglobulin fraction from ox serum was maintained at acid pH for several days and was then incubated at pH7.5 and 37 degrees . At least two polypeptides capable of stimulating smooth muscle were produced and attempts were made to obtain these in the pure state. 2. Only one substance could be purified completely and this was found to be a new plasma kinin, methionyl-lysyl-bradykinin. 3. The biological properties of this new kinin were compared with those of bradykinin and with a sample of synthetic methionyl-lysyl-bradykinin. 4. The possible significance of these results in relation to plasma-kinin formation by kallikrein and other enzymes is discussed.     

Identification of a novel conjugate in human urine: bile acid acyl galactosides

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.     

Identification of novel proteolytic forms of osteocalcin in human urine

In this study, we report the isolation and characterization of osteocalcin in human urine using mass spectrometry and N-terminal sequencing. Multiple proteolytic forms of osteocalcin were found, which consisted of 16-27 residues from the middle region of the molecule. Several fragments had residue Gly7 at the N-terminus and the most predominant was fragment 7-31. Additional fragments starting from residue Asp14 were detected in the samples of children and young adults. Immunochemical detection of urine osteocalcin fragments had a statistically significant negative correlation to bone mineral density in evaluation of urine samples from 75-year-old women. Thus, the measurement of osteocalcin fragments in urine may have potential applications in diagnostics related to disorders of bone metabolism.     

Identification of iso-TC-1 as a new T-2 toxin metabolite in cow urine

The structure of a new metabolite T-2 toxin (iso-TC-1) has been established as 3,15-diacetoxy-4-hydroxy-8(3-methyl-3'-hydroxy-butyryloxy)-12, 13-epoxytrichothec-9-ene. The compound is an isomer of TC-1 (a recently isolated T-2 derivative) in which the hydroxy and acetoxy groups at the C-3 and C-4 positions, respectively, are reversed. Direct probe analysis by electron impact (EI) of the underivatized iso-TC-1, as well as EI, positive chemical ionization (CI) in methane, and positive CI in ammonia of its trimethylsilylether or trifluoroacetate provided evidence to support the structure assignment of the new metabolite. The mass spectra of iso-TC-1 were compared with those of TC-1, T-2 toxin and iso-T-2 toxin (the isomer of T-2 toxin having reversed substituents at C-3 and C-4) with regard to molecular weight and fragments involving the substituents at C-3, C-4, C-8 and C-15. Although the two isomers, TC-1 and iso-TC-1, were not easily resolved by thin layer chromatography (TLC), a very good separation of their trimethylsilyl and trifluoroacetate derivatives was obtained by capillary gas chromatography. Acetylation of TC-1 or iso-TC-1 gave the same product. Iso-TC-1 is one of the main products of T-2 metabolism in the cow (more abundant than TC-1) and is found in the urine.     

Identification of 6-furfuryladenine (kinetin) in human urine

In contrast to the current view of kinetin (K, N(6)-furfuryladenine) as an unnatural and synthetic cytokinin, recently it has been identified in plant DNA and plant extract. Here we describe identification of K in human urine using chromatography/mass-spectrometry analysis for the first time. The amount of kinetin in urine taken from unhealthy patients lung carcinoma was established to be 0.5 ng in 20 ml and a 100-fold reduced amount in healthy subjects. Since this rare base is a potential source of structural constrains it has to be removed from DNA by enzymatic DNA-repair reactions. It seems that the presence of kinetin in human is linked to oxidative damage processes.     

Identification and quantitative determination of 2-hydroxyoestriol in human late-pregnancy urine

2-Hydroxyoestriol was isolated from human late-pregnancy urine and quantitatively determined by g.l.c. The purification procedure involved two chromatographic steps on silica gel columns and two paper Chromatographic separations using ascorbic acid impregnated stationary phases. The identity of the 2-hydroxyoestriol isolated was proved by gas chromatography-mass spectrometry and by a microchemical reaction. The urinary excretion of 2-hydroxyoestriol at the end of pregnancy, being in the order of 0.1 mg/24 h, was found to be lower than the conc. of 2-hydroxyoestrone in the same urine with only one exception.     

Identification and assay of tetranor-prostaglandin E1 in human urine.

7α, 11-Dihydroxy-5-keto-tetranorprost-9-enoic acid (tetranor-PGE_t) is a metabolite of PGE₂ and PGE₁ resulting from biotransformation only by β-oxidation. The preparation of pentadeutero-tetranor-PGE₁ is described, together with its use as a carrier-internal standard for the quantitative determination of tetranor-PGE₁. The method employs addition of a known amount of deuterated standard to the sample and, after its isolation and derivatization, measurement of the ratio of protium and deuterium forms by gas-liquid chromatography-mass spectrometry employing the technique of selected ion monitoring. Ratios of protium/D₅-tetranor-PGE₁ ranging from 0.006 to 0.084 (amount of the protium form per injection = approximately 0.3–4 ng) can be measured with a precision of ±6.5 to ±1.3% (SD), respectively, yielding a linear standard curve. Normal human females were found to excrete tetranor-PGE₁ in an average amount of 342 ng (±116) per day. A mass spectrum of this metabolite isolated from urine confirmed its identity.     

A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.

A glycopeptide, 3-O-beta-D-glucopyranosyl-alpha-L-fucopyranosyl-L-threonine, has been isolated from normal human urine. The glycopeptide was isolated by gel chromatography, preparative zone electrophoresis, paper chromatography, and high voltage electrophoresis. The average yield of the glycopeptide was in the range of 0.2 to 0.3 mg/liter of urine. Sugar analysis and amino acid analysis gave equimolar amounts of glucose, fucose, and threonine. Linkages and sequential order were established by methylation analysis of the glycopeptide after degradation of the amino acid residue with ninhydrin. The permethylated product was analyzed on gas liquid chromatography and mass spectrometry. Anomeric configuration was deduced from optical rotation.