Urinary D-asparagine level is decreased by the presence of glioblastoma.

Gliomas, particularly glioblastomas (GBMs), pose significant challenges due to their aggressiveness and poor prognosis. Early detection through biomarkers is critical for improving outcomes. This study aimed to identify novel biomarkers for gliomas, particularly GBMs, using chiral amino acid profiling. We used chiral amino acid analysis to measure amino acid L- and D-isomer levels in resected tissues (tumor and non-tumor), blood, and urine from 33 patients with primary gliomas and 24 healthy volunteers. The levels of D-amino acid oxidase (DAO), a D-amino acid-degrading enzyme, were evaluated to investigate the D-amino acid metabolism in brain tissue. The GBM mouse model was created by transplanting GBM cells into the brain to confirm whether gliomas affect blood and urine chiral amino acid profiles. We also assessed whether D-amino acids produced by GBM cells are involved in cell proliferation. D-asparagine (D-Asn) levels were higher and DAO expression was lower in glioma than in non-glioma tissues. Blood and urinary D-Asn levels were lower in patients with GBM than in healthy volunteers (p < 0.001), increasing after GBM removal (p < 0.05). Urinary D-Asn levels differentiated between healthy volunteers and patients with GBM (area under the curve: 0.93, sensitivity: 0.88, specificity: 0.92). GBM mouse model validated the decrease of urinary D-Asn in GBM. GBM cells used D-Asn for cell proliferation. Gliomas induce alterations in chiral amino acid profiles, affecting blood and urine levels. Urinary D-Asn emerges as a promising diagnostic biomarker for gliomas, reflecting tumor presence and severity.

Urinary l-erythro-ß-hydroxyasparagine-a novel serine racemase inhibitor and substrate of the Zn2+-dependent d-serine dehydratase.

In the present study, we identified l-erythro-ß-hydroxyasparagine (l-ß-EHAsn) found abundantly in human urine, as a novel substrate of Zn2+-dependent d-serine dehydratase (DSD). l-ß-EHAsn is an atypical amino acid present in large amounts in urine but rarely detected in serum or most organs/tissues examined. Quantitative analyses of urinary l-ß-EHAsn in young healthy volunteers revealed significant correlation between urinary l-ß-EHAsn concentration and creatinine level. Further, for in-depth analyses of l-ß-EHAsn, we developed a simple three-step synthetic method using trans-epoxysuccinic acid as the starting substance. In addition, our research revealed a strong inhibitory effect of l-ß-EHAsn on mammalian serine racemase, responsible for producing d-serine, a co-agonist of the N-methyl-d-aspartate (NMDA) receptor involved in glutamatergic neurotransmission.

Altered glycosylation is induced in both alpha- and beta-subunits of human chorionic gonadotropin produced by choriocarcinoma.

The two subunits of human chorionic gonadotropin (hCG) purified from the urine of a patient with choriocarcinoma were successfully separated by SDS-polyacrylamide gel electrophoresis. A comparative study of the oligosaccharides released from the two subunits by hydrazinolysis revealed that altered glycosylation occurs in both subunits and possibly at all four asparagine sites of the choriocarcinoma hCG molecule.

Characterization of two glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU).

Two major glycoasparagines (2-acetamido-N-(4'-L-aspartyl)-2-deoxy-beta-D-glycosylamines) were isolated from the urine of patients with aspartylglycosylaminuria (AGU). They were composed of equimolar amounts of sialic acid, galactose, glucosamine, and aspartic acid. They were isomeric with respect to the position of sialic acid attachment, since they produced the same glycoasparagine on incubation with the neuraminidase from Clostridium perfringens. The structure of the resulting sialic acid-free glycoasparagine was determined as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based on the following findings. It produced galactose on incubation with beta-galactosidase, and N-acetyllactosamine and aspartic acid on incubation with 4-L-aspartylglycosylamine amindo hydrolase.

Characterization of one neutral and two acidic glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU).

One neutral and two acidic glycoasparagines were isolated from the urine of patients with aspartylglycosylaminuria (AGU). The neutral one was identified as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn. The acidic ones were composed of 1 mole of sialic acid and 2 moles each of galactose and N-acetylglucosamine, attached to asparagine, and were isomeric with respect to the position of sialic acid attachment since they produced the same glycoasparagine on incubation with the neuraminidase [EC 3.2.1.18] from Clostridium perfringens. The structure of the resulting sialic acid-free glycoasparagine was determined to be beta-Gal-beta-GlcNAc-beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based mainly on the results of sequential enzymatic degradations.

Structure of two glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU).

The structures of two glycoasparagines composed of one mole each of N-acetylneuraminic acid, galactose, N-acetylglucosamine, and asparagine were determined by periodate oxidation, enzymatic degradation, and methylation analysis. The structures were NANAalpha2 leads to 3Galbeta1 leads to 4GlcNAcbeta leads to Asn and NANAalpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta leads to Asn, respectively.

Characterization of beta-galactosidase from a special strain of Aspergillus oryzae.

beta-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from alpha-galactosidase, alpha- and beta-mannosidase, alpha- and beta-N-acetylhexosaminidase, and protease activities. The beta-galactosidase was capable of acting on aryl beta-galactosides, lactose, and lactosides. It also hydrolyzed beta-galactosyl linkages in urinary glycoasparagines and asialo alpha1-acid glycoprotein. The enzyme was rather stable in aqueous solution, retaining full activity at 4 degrees for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37 degrees, full activity was maintained for several days.

Identification of 4-N-2-acetamido-2-deoxy-beta-D-glucopyranosyl-L-asparagine in biological materials by gas chromatography-mass spectrometry.

A specific and sensitive method for the identification of 4-N-2-acetamido-2-deoxy-beta-D-glucopyranosyl-L-asparagine (GlcNAc-Asn) in urine in aspartylglycosaminuria and in hydrolysates of glycoproteins is described. The method involves permethylation of GlcNAc-Asn followed by gas chromatographic-mass spectrometric analysis of the methylated derivative. It can be used to confirm the diagnosis of aspartylglycosaminuria and to assess the excretion of GlcNAc-Asn in urine during various phases of the disease. The presence of an N-acetylglucosaminyl-asparagine type of carbohydrate-peptide linkage in a glycoprotein can be determined by applying the method to the partial acid hydrolysate of a proteolytically digested glycoprotein.

Measurement of urinary amino acids using high-performance liquid chromatography equipped with a strong cation exchange resin pre-column.

We determined the urinary amino acid concentrations by high-performance liquid chromatography (HPLC) using an eluent buffer which had been processed through a column packed with strong cation-exchange resin. With this column, ghost peaks resolved or much reduced in size and shifted to a post-arginine position. A clearer separation of peaks of urinary amino acids and ammonia was obtained. This could be due to the elimination of O-phthalaldehyde reactive substances in the eluent by the resin column. This method does not require a separate step to remove ammonia, unlike most of the methods currently used, and thus saves time.

Urinary abnormalities in fucosidosis. Characterization of a disaccharide and two glycoasparagines.

The urinary excretion of fucose-containing material was found to be highly increased in a patient with fucosidosis type 2. Three structurally related compounds, a disaccharide and two glycoasparagines, were isolated from the urine. The isolation procedure included ultrafiltration, gel chromatography on Sephadex G-25, preparative zone electrophoresis and paper chromatography. From structural studies including optical rotation, sugar analysis, methylation analysis, ninhydrin degradation, reduction with lithium aluminium hydride and partial hydrolysis, the following structures were deduced: formula (see text), where Fucp is fucopyranose, Manp is mannopyranose, Galcp is galactopyranose, GlcNAcp is 2-acetamido-2-deoxyglucopyranose and Asn is asparagine. The yields of these compounds were 1.7, 40, and 6 mg/l, respectively. The origin of the disaccharide and the two glycoasparagines is probably the core region of glycoprotein carbohydrate chains.