Amount Of N-acetylneuraminic Acid Research Articles

DMTMM-mediated methylamidation for MALDI mass spectrometry analysis of N-glycans with structurally conserved sialic acid residues in biological fluids "via direttissima".

Characterization of serum glycoprotein N-glycans with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in positive-ion mode needs a derivatization step to stabilize and neutralize the negative charge on sialic acids. The acidic sugars are attached to the end of glycoproteins, glycolipids or gangliosides. Here, we present a method for sialic acid stabilization via modification based on derivatization of carboxylic acid group activated with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) with methylamine. DMTMM substitutes in many processes N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide (EDC/NHS) as activation reagent due to its better performance and higher stability in water. Glycosylated proteins are used as solid phase support for glycan derivatization and purification from excess of derivatization reagents. We evaluated our glycan analysis method in murine sera and intestinal lavages. The stabilization of sialic acid enables a complete conservation of the glycan structures, in contrast to other methods where sialic acids are partially lost. In BALB/c mouse sera, we detected predominantly mono- and di-sialylated N-glycans with mostly N-Glycolylneuraminic acid (Neu5Gc) and only trace amounts of N-Acetyl neuraminic acid (Neu5Ac). BALB/c mouse intestinal lavages glycoproteins contained asialo N-glycans. DMTMM-mediated methylamidation of N-glycans for MALDI mass spectrometry analysis is a fast and cheap method for structurally conserved glycan derivatization.

Letter to the Editor regarding “Lactose does not interfere with the analysis of sialic acids as their 1,2-diamino-4,5-methylenedioxybenzene derivatives”

Dear Sir,Recently, Spichtig et al. [1] published in this journal aTechnical Note regarding the analysis of sialic acids frominfant formulas using 1,2-diamino-4,5-methylenedioxyben-zene derivatives, a method previously proposed by us in2007 [2]. In this Technical Note, Spichtig et al. stated thatlactose does not interfere with the analysis, conversely towhat we hypothesized in our article. Although in the workreported in their Technical Note they did not include infantformulas or milk, in workpublished in 2010 [3]they appliedthe proposed method to several dairy products, includinginfant formulas. In the most recent work, they used com-mercial lactose and quantified fetuin sialic acids when thisprotein was in lactose solution (no concentration reported)and in aqueous solution. They reported an increase of30 μg/100 mg in the former solution when comparedwith the latter, which they attributed to a certain amountof sialyllactose present in the commercial lactose be-cause of its dairy origin. This residual sialic acid in lactosewas quantified (6.5-24.7 μg/100 mg), and the concentrationdiffered withthe supplierand was consideredverylow bytheauthors.Regarding this controversial topic, and in response to theaforementioned article, we would like to clarify our previousreport.First,weobservedaproblemintheaccuratequantitationof sialic acids from infant formulas and proposed a way toeffectively resolve this via an additional purification step. Wesuggested that lactose could be a possible reason for the ob-served phenomenon. In fact, in a series of recent tests in ourlaboratory, when commercial lactose (from Sigma) was spikedwith different amounts of N-acetylneuraminic acid (Neu5Ac)and quantified using the method reported the recovery in-creased as the amount of Neu5Ac added increased: 5.15 μgwas recovered when 5 μg was added, but 19.55 μg wasrecoveredwhen15μg was added. This represents a matrixeffect caused by lactose that produces a systematic error pro-portional to the concentration of Neu5Ac. So, we do not agreewith the statement in the abstract of the technical note thatlactose does not interfere with the analysis and we stronglyrecommend the purification step if accurate quantitation ofsialic acids in infant formulas is required.References

Effect of Vitamin A Palmitate on the Synthesis of Mucins in Cultured Conjunctiva

Purpose: To evaluate the effect of vitamin A palmitate on the synthesis of mucins from goblet cells in cultured rabbit conjunctiva. Methods: Palpebral conjunctiva was obtained from 12 white rabbits and was cultured in serum-free RCGM 2 which was developed for culture of rabbit corneal epithelial cells. This method eliminated retinol in the serum. High-performance liquid chromatography was used in quantitating N-acetyl-neuraminic acid contained in the sugar chain as an indicator of mucin content. Results: Addition of vitamin A palmitate to the cultured medium resulted in significant increase in the amount of N-acetylneuraminic acid in the medium. Conclusions: The increase in N-acetylneuraminic acid in the cultured medium suggests that the increase of goblet cells in cultured conjunctiva is related to the synthesis of mucins due to vitamin A. RCGM 2 as a culture medium promises to be useful in evaluating the pharmacological activity of vitamin A. High-performance liquid chromatography facilitated quantitation of mucins in the culture medium.

N-Acetylneuraminic acid and N-glycolylneuraminic acid in the O-linked oligosaccharides of a tumor cell glycoprotein. Incorporation and distribution.

The MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma, which differ in several cell surface properties, contain a major mucin-type glycoprotein, termed ASGP-1. The sialic acid content of MAT-C1 ASGP-1 is 2-3-fold greater than MAT-B1 ASGP-1 (Sherblom, A. P., Buck, R. L., and Carraway, K. L. (1980) J. Biol. Chem. 255, 783-790). Sialic acid analysis demonstrated that, whereas MAT-C1 ASGP-1 contained approximately equal amounts of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGl), MAT-B1 ASGP-1 was devoid of NeuGl. MAT-B1 microsomes also did not contain NeuGl. MAT-B1 cells incubated with [3H]N-acetylmannosamine did not synthesize either labeled CMP-NeuGl or free NeuGl, even though the CMP-sialic acid synthetase was active with the substrate NeuGl. Thus, MAT-B1 cells may be deficient in the enzyme N-acetylneuraminate monooxygenase. The O-linked oligosaccharides from both MAT-B1 and MAT-C1 ASGP-1 have been shown to contain a core tetrasaccharide Gal(beta 1-4)GlcNAc(beta 1-6)(Gal(beta 1-3]GalNAc in which both galactose residues may be linked to additional sugars (Hull, S. R., Laine, R. A., Kaizu, T., Rodriquez, I., and Carraway, K. L. (1984) J. Biol. Chem. 259, 4866-4877). The distribution of NeuAc and NeuGl between the two galactose termini of the core tetrasaccharide was examined for MAT-C1 ASGP-1. Oligosaccharides were released by alkaline-borohydride treatment of MAT-C1 ASGP-1 which had been labeled with [14C]glucosamine and galactose oxidase/B3H4. Following fractionation by Bio-Gel P-4, DEAE-Sephadex, and high-performance liquid chromatography, oligosaccharides were analyzed for NeuAc and NeuGl and for susceptibility to digestion with beta-galactosidase. Three disialylated oligosaccharides were identified containing 2 mol of NeuAc (5.5% recovery), 2 mol of NeuGl (4.5%), or 1 mol each of NeuAc and NeuGl (11.1%). For monosialylated oligosaccharides, NeuGl appeared preferentially associated with the Gal(beta 1-4)GlcNAc terminus (9.0%), whereas significant amounts of oligosaccharide containing NeuAc at both the Gal(beta 1-3)GalNAc (2.6%) and Gal(beta 1-4)GlcNAc (4.5%) termini were detected. Each of the major qualitative differences between MAT-B1 and MAT-C1 oligosaccharides, including the presence of NeuGl (MAT-C1), sulfate (MAT-B1), and alpha-linked galactose (MAT-B1), occurs at the Gal(beta 1-4)GlcNAc terminus.

Sialic acids in epithelial tissues of Carassius auratus gibelio (bloch) (prussian carp), Cottus gobio L. (bullhead) and Myoxocephalus scorpius (L.) (bull rout)

1. 1. Homogenates of tissues which participate in osmoregulation of fishes were assayed for N-acetyl-neuraminic acid (NANA). 2. 2. The kidneys were found to contain considerable amounts of NANA, in addition to well known sites of sialoglycoprotein production (e.g. gills, intestine, skin). 3. 3. The epithelia of freshwater fish ( Carassius auratus, Cottus gobio) exhibited higher concentrations of NANA as compared with saltwater fish ( Myoxocephalus scorpius). 4. 4. The biochemical results are discussed, for their functional significance.

Effect of human cerebral neuraminidase on hexosaminidase A

Hexosaminidase A from liver or placenta can be converted to an enzyme that behaves like hexosaminidase B by either bacterial or cerebral neuraminidase preparations. Concurrent with this change increasing amounts of N-acetyl neuraminic acid are released into the reaction mixture. Therefore, two properties of hexosaminidase isozymes, heat lability and electrophoretic migration, are due to N-acetyl neuraminic acid content. This interconversion may have metabolic significance.

Memory consolidation and glycoprotein metabolism: A failure to find a relationship

In an effort to study the role of glycoproteins in memory consolidation and stress, albino rats were trained for 3–5 minutes on a simple one trial passive avoidance of foot shock task. A three minute retention test was run 24 hours after original learning. [1− 14C] Glucosamine (15 microcuries/rat) was injected intraperitoneally 15 minutes before original learning (Experiments 1 and 2), before retention (Experiment 3), or before 10 minutes of inescapable foot shock (Experiment 4). Control animals were handled in the same fashion as experimental subjects, except that the former never received foot shock. Sacrifice occured one hour (Experiments 1,3 and 4) or twenty four hours (Experiment 2) after injection. There were no significant differences between controls and experimental subjects in the incorporation of isotope into the heteropolysaccharide chains isolated from brain glycoproteins. The amount of N-acetylneuraminic acid (NANA), hexose, hexosamine, and fucose in the heteropolysaccharide chains were also unchanged. The view that glycoproteins are involved in memory received no support from the present findings.

Teneur en caséine κ de la caséine de colostrum de vache

SummaryCasein derived from cow's colostrum contains 0·85%N-acetyl neuraminic acid and liberates 2·95% nitrogen soluble in 12% trichloroacetic acid (NPN) under the action of rennin. It therefore appears to contain a large proportion of κ-casein (calculated to be approximately 35%).The smaller the micelles of casein in colostrum the larger is the amount ofN-acetyl neuraminic acid they contain. Soluble casein, i.e. 25% of the casein in colostrum, contains 1·52% of this acid and gives 6·24% NPN under the action of rennin.Casein and particularly the soluble casein of colostrum appear to be important sources of κ-casein and caseino-glycopeptide.

Teneur en acide N-acétylneuraminique de sérums sanguins humains

1. (1) Numerous papers are cited that deal with sialic acids in various biological materials and that are not included in the reviews on this subject mentioned here. 2. (2) Using the technique of Svennerholm-Miettinen and Takki-Luukkainen the content of N-acetylneuraminic acid (NANA) was determined in 179 sera from 161 normal and pathological individuals. 3. (3) The mean value of the NANA content in 25 normal subjects (from 22–27 years of age) was 60 ± 10.4 mg/100 ml sera, which agrees fairly well with the averages given by some of the authors mentioned. 4. (4) In 27 patients with tuberculosis at various stages, the average was found to be 87.4 ± 20 mg/100 ml; in 21 children with rachitis, this value was 77.7 ± 16.5 mg/100 ml before treatment; after treatment with vitamin D 3 the values decreased considerably. 5. (5) In normal individuals the NANA content varies with age: in 5 new-born babies values of 39.1 ±5.8 mg/100 ml were found, which is lower than those obtained for 7 children aged from 4 months to 2 years (64.4 ± 74 mg/100 ml). A few hours before giving birth the mothers of these new-born babies had higher values than those considered as normal. Further data on this subject are being collected. 6. (6) In 31 patients (under 11 years of age) suffering from various diseases and with low red cell counts (3.6 ± 1 million/mm 3 blood), a considerable increase in the amount of NANA in the serum was found.