Concentration Of N-acetylneuraminic Acid Research Articles

Elevated concentrations of Neu5Ac and Neu5,9Ac2 in human plasma: potential biomarkers of cardiovascular disease

Cardiovascular disease (CVD) is a group of health conditions affecting the heart and vascular system with very high prevalence and mortality rates. The presence of CVD is characterised by high levels of inflammation which have previously been associated with increased plasma concentrations of N-acetyl neuraminic acid (Neu5Ac). While Neu5Ac has been studied in the context of CVD, Neu5,9Ac2 has not, despite being the second most abundant sialic acid in human plasma. A small-scale pilot study of thirty plasma samples from patients with diagnosed CVD, and thirty age and sex-matched healthy controls, was designed to gain insight into sialic acids as biomarkers for CVD and potential future areas of study. Each sample was assayed for Neu5Ac and Neu5,9Ac2 concentrations. Mean Neu5Ac and Neu5,9Ac2 concentrations were significantly elevated in patients with CVD compared to healthy controls (Neu5Ac: P < 0.001; Neu5,9Ac2: P < 0.04). Receiver operator curve (ROC) analysis indicated that both Neu5Ac and Neu5,9Ac2 have reasonable predictive power for the presence of CVD (Neu5Ac AUC: 0.86; Neu5,9Ac2 AUC: 0.71). However, while Neu5Ac had both good sensitivity (0.82) and specificity (0.81), Neu5,9Ac2 had equivalent specificity (0.81) but very poor sensitivity (0.44). A combination marker of Neu5Ac + Neu5,9Ac2 showed improvement over Neu5Ac alone in terms of predictive power (AUC: 0.93), sensitivity (0.87), and specificity (0.90). Comparison to a known inflammatory marker, high sensitivity c-reactive protein (hs-CRP: P-value: NS, ROC:0.50) was carried out, showing that both Neu5Ac and Neu5,9Ac2 outperformed this marker. Further to this, hs-CRP values were combined with the three different sialic acid markers to determine any effect on the AUC values. A slight improvement in AUC was noted for each of the combinations, with Neu5Ac + Neu5,9Ac2 + hs-CRP giving the best AUC of 0.97 overall. Thus, Neu5Ac would appear to offer good potential as a predictive marker for the presence of CVD, which the addition of Neu5,9Ac2 predictive power improves, with further improvement seen by the addition of hs-CRP.

Milk Polysialic Acid Levels Rapidly Decrease in Line with the N-Acetylneuraminic Acid Concentrations during Early Lactation in Dairy Cows.

Sialylated milk oligosaccharides and glycoconjugates have several positive effects on the mucosal barrier, the gut microbiome, and an effective immune system. For this reason, they are important biomolecules for mammary gland health and optimal development of offspring. In milk, the major sialic acid, N-acetylneuraminic acid (Neu5Ac), can be attached as monosialyl-residues or as polymers. To investigate the sialylation processes during lactation of German Holstein cows, we analyzed udder tissue in addition to milk at different time points of lactation. The analysis of the milk samples revealed that both the levels of Neu5Ac and its polymer, polysialic acid (polySia), rapidly decreased during the first three days of lactation, and a high interindividual variance was observed. In mature milk, however, the sialylation status remains relatively constant. The results indicate that mammary gland epithelial cells are one source for milk polySia, since immunohistochemistry of udder tissue exhibited strong polySia staining in these cells. Furthermore, both polysialyltransferases, ST8SiaII and ST8SiaIV, are expressed. Based on known functions of monosialyl residues and polySia, we discuss the potential impact of these biomolecules and the consequences of the heterogeneous sialylation status of milk in relation to udder health and offspring health.

THE EFFECT OF S-ADEMETIONINE ON PLASMA CITRULLINE LEVEL DURING CHEMOTHERAPY-INDUCED OXIDATIVE STRESS IN PATIENTS WITH CHRONIC LYMPHOPROLIFERATIVE DISORDERS.

The aim: To investigate the effect of S-ademetionine on plasma citrulline level in patients with chronic lymphoproliferative disorders (CLPD) during chemotherapy-induced oxidative stress. Materials and methods: 25 patients with CLPD were examined. Examinations were conducted twice: before chemotherapy (CT) and after 3 courses of CT. Several biochemical markers in the blood were determined: the activity of catalase, the level of plasma citrulline, the concentration of N-acetylneuraminic acid (NANA) and the concentration of substances that form a trimethine complex (TBARS) with 2-thiobarbituric acid. Patients were divided into groups: І (n=14) - patients who underwent only CT; ІІ (n=16) - patients who during CT received S-ademetionine, at a dose of 1000 mg/day intravenously for 10 days, then 500 mg twice a day for 20 days. ІІІ (n=20) -the control group of 20 practically healthy individuals. Results: Patients in both groups with CLPD had pre-existed mucosal injury that was characterized by 1.25 (p=0.0025) and 1.26 times (р=0.006) higher blood NANA concentration compared to the control group. The conduction of CT was associated with enterocytes dysfunction, which was characterized by 1,66 times (p=0,0002) lower plasma citrulline level in patients of group I compared to the initial examination. The infusion of S-ademetionine attenuated intestinal dysfunction that was associated with 1,23 times (p=0,0005) higher blood citrulline level after the CT as compared to group I. Conclusions: The infusion of S-ademetionine as adjuvant treatment in patients with CLPD provided effective prophylaxis of intestinal injury that was associated with higher blood citrulline level after the conduction of CT.

Relationship Between Pathogenetic Mechanisms of NSAID-gastropathy Development in Elderly Patients with Concomitant Ischemic Heart Disease

The objective: to study the relationship between risk factors and mechanisms of NSAID gastropathy development with concomitant ischemic heart disease.Materials and methods. The study included 125 patients who underwent general clinical examination, upper endoscopy with biopsy of the gastric mucosa (GM), followed by histological examination of biopsies, assessment of mucosal function of GM (content of N-acetylneuraminic acid (NANA) and fucoproteins in the blood), oxidative stress (concentration of TBA-active products and catalase activity in the blood serum), endothelial dysfunction (nitrite concentration and endothelial NO-synthase (eNOS), inducible NOS (iNOS) activity), severity of erosive-ulcerative lesions according to modified Lanzascore scale with assessment of histological changes.Results. In most patients of both groups, the onset of complaints was provoked by taking ASA and/or NSAIDs and had a direct correlation with the number of anti-inflammatory drugs (r=0.72; r=0.63; p=0.025), and also depended on the duration of NSAIDs treatment (r=0.52; r=0.67; p=0.031). There is a strong direct relationship between the concentration of NANA in the blood serum and the amount of NSAIDs (r=0.69; p=0.03), the duration of drugs (r=0.50; p=0.024), the content of TBA reactants in the blood serum (r=0.59; p=0.015), the severity of erosive-ulcerative lesions according to Lanzascore scale (r=0.71; p=0.017) on the background of inverse correlation with eNOS activity (r=–0,65; р=0,024). Correlation analysis showed a direct relationship between iNOS activity and H. pylori infection (r=0.63; p=0.030), the severity of erosive-ulcerative lesions according to Lanzascore scale (r=0.50; p=0.047), the GM infiltration by polymorphonuclear leukocytes (r=0.72; p=0.027) against the background of inverse correlation with the catalase activity (r=–0.41; р=0.030).Conclusion. The degree of GM injury depends on the amount and duration of NSAIDs and/or ASA, the presence of comorbid pathology, H.pylori infection, oxidative stress activity, endothelial dysfunction.

SYNERGISTIC EFFECT OF QUERCETIN AND EPIGALLOCATECHIN-3-GALLATE AS AGENTS TO PREVENT CONNECTIVE TISSUE DISINTEGRATION IN THE PERIODONTIUM OF RATS UNDER SYSTEMIC AND LOCAL ADMINISTRATION OF LIPOPOLISACCHARIDE OF SALMONELLA TYPHI

The aim of the present study was to investigate the co-effect produced by water-soluble form of quercetin and epigallocatechin-3-gallate (EGCG) on biochemical markers of periodontal organic matrix depolimerization under systemic administration and local application of S. typhi lipopolisaccharide (LPS). The studies were conducted on 30 white rats of the Wistar line weighing 180-220 g, divided into 5 groups: the 1st included intact animals, the 2nd was made up of animals after the combined systemic and local LPS administration, the 3rd and 4th groups included animals, which were being given injections with water-soluble form of quercetin (10 mg / kg) and EGCG (21.1 mg / kg) respectively 3 times a week, starting on the 30th day of the systemic LPS administration, and the 5th group involved rats, which were injected with co-administered water-soluble form of quercetin and EGCG. It has been found out that the co-effect produced by quercetin and EGCG under systemic and local LPS administration is accompanied with reduced concentration of N-acetylneuraminic acid (NANA) by 31.8 and 32.8% respectively in the soft periodontal tissues compared with values for the animals received separate quercetin and EGCG during the experiment. However, no differences have been detected between the groups exposed to combined or separate action of the above mentioned agents in the experiment when assessing free hydroxyproline (FHP) and glycosaminoglycans (GAGs) content in the soft tissues of periodontium. At the same time combined use of quercetin and EGCG under experimental conditions led to the decrease in the FHP content in the alveolar bone by 24.5 and 20.2% respectively compared with values for the animals received separate quercetin and EGCG. NANA concentration was reduced by 35.0 and 41.3% respectively. Thus, the co-administration of water-soluble form of quercetin and epigallocatechin-3-gallate under systemic and local introducing of S. typhi lipopolysaccharide has been proven to be more effective means for preventing and correcting periodontal connective tissue disruption than this occurs at separate administration of each of the polyphenols.

Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method

The measurement of urine sialic acid (N Acetylneuraminic Acid: Neu5Ac) is useful for screening sialic acid storage disorders. We developed a new LC MS/MS method for the determination of a sialic acid. Urine samples were analyzed, after an HCl n-Butanol derivatization step, by a reverse phase based high-performance liquid chromatography method using 1,2,3- 13C 3 N-Acetyl- d-neuraminic Acid ( 13C-Neu5Ac) as an internal standard. Selective detection was performed by tandem mass spectrometry using an electrospray source operating in positive ionization mode employing multiple reactions monitoring to monitor N-Acetylneuraminic Acid and the internal standard. The transitions m/ z 366 → 330 and 369 → 333 for Neu5Ac and 13C-Neu5Ac were respectively monitored. The limit of the method quantification was 1.40 μM of N-Acetylneuraminic Acid and the calibration curve showed a good linearity up to 1000 μM. The within assay precision and accuracy of the method ranged from 3.22 to 5.95% and 98.69 to 109.18%, respectively and the between assay precision and accuracy ranged, respectively, from 5.15 to 7.65% and 96.14 to 102.30%. The method can be applied for the determination of N-Acetylneuraminic Acid concentrations in urine and other biological fluids (e.g., amniotic and peritoneal fluids).

Isolation of N-acetylneuraminic Acid and N-glycolylneuraminic Acid from Pleurocybella porrigens

Pleurocybella porrigens (P. porrigens) is a traditional food consumed in Japan. Toxicity was first reported in 2004, following which a series of poisonings were reported in 2007. More than 59 people who consumed P. porrigens suffered from similar severe cryptogenic encephalitis, with an overall death rate of approximately 29%. P. porrigens is believed to be a major etiological agent of this disease, but the mechanism of pathogenesis is not clear. To elucidate the toxic properties of P. porrigens in the 2004 and 2007 poisonings, we compared the oligosaccharide constituents of mushroom samples collected in these years with those collected in other years. Water extracts (90°C and 4°C) of P. porrigens were dialyzed, and the oligosaccharides obtained from the high-molecular-weight fraction (>7.8 kDa) were subjected to acid hydrolysis for modification and labeling. Resultant saccharides were analyzed by high performance liquid chromatography on an octadecyl silane (ODS) column. Our analysis revealed that the concentration of N-acetylneuraminic acid (NeuAc) was abundant in all samples, however, N-glycolylneuraminic acid (NeuGc) was present only in significant amounts in the P. porrigens samples collected in 2004 and 2007.

N-acetylneuraminic acid (NANA) stimulates in situ cyclic AMP production in tentacles of sea anemone (Aiptasia pallida): possible role in chemosensitization of nematocyst discharge.

Cnidocytes, the stinging cells of cnidarians, optimally discharge nematocysts in response to combined physical contact and stimulation of specific chemoreceptors. In the tentacles of certain sea anemones, the primary chemoreceptors bind N-acetylated sugars, such as N-acetylneuraminic acid (NANA). Sensitization with NANA predisposes contact-sensitive mechanoreceptors (CSMs) to trigger discharge in response to physical contact. In the ectoderm of sea anemone tentacles, cnidocyte/supporting cell complexes (CSCCs) control and trigger nematocyst discharge. Previous findings have implicated cyclic AMP (cAMP) as a second messenger in NANA-sensitized nematocyst discharge. However, no reports have directly demonstrated that the cAMP content of tentacles changes in response to NANA stimulation. We now show that NANA elevates in situ cAMP levels in a dose-dependent manner in the ectoderm of tentacles from the sea anemone Aiptasia pallida. However, the endoderm of tentacles shows no detectable cAMP response to NANA. The effect of NANA on the cAMP content of the ectoderm is biphasic. Micromolar NANA increases the in situ cAMP level, with a maximal response occurring at 1.8x10(-5)mol x l(-1) NANA. At higher NANA concentrations, the cAMP content decreases to that of controls. Because the cAMP dose/response curve to NANA coincides precisely with the dose/response curves of NANA-sensitized nematocyst discharge and nematocyst-mediated adhesive force, a second-messenger role for cAMP in NANA-sensitized nematocyst discharge is strongly suggested. The addition of isobutyl-1-methylxanthine (IBMX) to the medium with sea anemones increases tissue cAMP levels both in the absence and in the presence of NANA. However, anesthetizing anemones in sea water containing high levels of Mg(2+) blocks the NANA-stimulated cAMP response of the ectoderm. In addition, our results suggest that NANA-stimulated cAMP may activate endogenous cAMP-dependent protein kinase (PKA) in broken cell preparations of tentacles. Thus, NANA-stimulated cAMP may function as a second messenger in the NANA chemosensory signaling pathway controlling nematocyst discharge.

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 umol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 umol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Inhibitory effect of free sialic acid on complement activation and its significance in hypocomplementemic glomerulonephritis

The role of free sialic acid on complement activation was investigated. The serum levels of free sialic acid and total sialic acid were measured by previously described methods in 16 patients with acute post-infectious glomerulonephritis (AGN), 27 patients with systemic lupus erythematosus (SLE), 15 patients with persistent hypocomplementemic membranoproliferative glomerulonephritis (MPGN), and 13 healthy controls. A statistical study demonstrated an increased level of free sialic acid in patients with AGN and SLE in which the hypocomplementemia improved throughout the course and a decreased level of free sialic acid in patients with MPGN and SLE in which hypocomplementemia continued throughout the course. The levels of total sialic acid were significantly increased in patients with AGN and SLE and were significantly decreased in patients with MPGN. There was no correlation between the levels of free sialic acid and total sialic acid in patients with AGN, in whom the levels of both total and free sialic acids were increased. To examine the effect of free sialic acid on the complement cascade, lipopolysaccharide (LPS) was incubated with normal human serum (NHS) in the various concentrations of N-acetyl neuraminic acid (NANA), a member of the sialic acid group. The incubation mixtures were examined by enzyme immunoassay using monoclonal anti-iC3b antibody or anti-Bb antibody. Native C3 or Factor B in NHS broke down less following the addition of NANA. To elucidate the role of NANA on the hemolytic function of C3, a rabbit erythrocyte (Ra E) hemolytic assay was carried out. Ra E lysed completely in the presence of R3 with native C3. However, hemolysis occurred to a lesser degree in C3-depleted serum (R3) or R3 with NANA-treated C3. To investigate the influence of NANA on complement components, the levels of complement components were measured in the incubation mixture with various doses of NANA and NHS. The levels of C3 and C5 were significantly decreased after the addition of NANA, even though the levels of Factor H and Factor I were not markedly changed. These data indicate that NANA exerts an influence on the complement components even though it has no effect on the regulatory proteins of complement. Our in vitro findings, together with the in vivo data, suggest that free sialic acid might have an inhibitory effect on the activation of C3 and the following complement cascade, and might also have been responsible for the improvement of hypocomplementemia.

Chronic administration of propionic acid reduces ganglioside N-acetylneuraminic acid concentration in cerebellum of young rats.

Elevated levels of propionate comparable to those of human propionic acidaemia were achieved in the blood of young rats by injecting subcutaneously buffered propionic acid (PPA) twice a day at 8-h intervals from the 6th to the 28th day of life. A matched group of animals (controls) was treated with the same volumes of saline. The animals were weighed and sacrificed by decapitation at 28, 35 or 60 days of age. Cerebellum and cerebrum were weighed and their protein and ganglioside N-acetylneuraminic acid (G-NeuAc) contents determined. Body, cerebral and cerebellar weights were similar in both groups, suggesting that PPA per se neither alters the appetite of the rats nor causes malnutrition. Brain protein concentration was also not affected by chronic administration of PPA, in contrast to G-NeuAc concentration which was significantly reduced in the cerebellum. Since ganglioside concentration is closely related to the dendritic surface and indirectly reflects synaptogenesis, our results of an important ganglioside deficit in the brain of PPA-treated animals may be related to the neurologic dysfunction characteristic of propionic acidaemic patients.

Single-step isolation method for six glycoforms of human α 1-acid glycoprotein by hydroxylapatite chromatography and study of their binding capacities for disopyramide

A single-step isolation method for the glycoforms of human serum α 1-acid glycoprotein (AAG) using a hydroxylapatite column under a gradient elution program was developed. The concentrations of N-acetylneuraminic acid and monosaccharides (fucose, N-acetylglucosamine, galactose and mannose) of six AAG glycoforms were determined by the pulsed-amperometric detection method. For each AAG glycoform, significant sex-related differences in carbohydrate content have been observed only for AAG glycoforms two and six, and not for each AAG glycoform. The relationship between the extent of the branch in the glycan chain and the binding capacity to disopyramide were examined. Female AAG contained highly sialylated AAG glycoforms compared to male glycoforms. Conversely, male AAG was rich in the lower sialylated AAG glycoform. Furthermore, it was found that the drug binding capacity decreases with increasing branching of the glycan chain. This suggests that the binding sites of AAG are hindered by a relatively large carbohydrate moiety, such as tetraantennary structures.

Evaluation of tumor-associated antigens and total sialic acid levels in effusions of malignant and benign origin.

Cytological examination is the most commonly used diagnostic procedure in the differential diagnosis of pleural effusions although the sensitivity of this method in malignancy compared with more invasive procedures is only about 60 'YO Several studies have tried to increase the sensitivity of the cytological examination by using additional immunological or serological methods such as the determination of lactate dehydrogenase, total protein content, tumor-associated antigens, DNA aneuploidea. or expression of different cellular antigens. While the analysis of cellular parameters requires some technical expertise, the detection of humoral factors is more simple. Especially tumor-associated antigens, released by the tumor cells into the effused fluid could possibly become significant in the diagnostic differentiation of the metastatic from the nonmetastatic or benign origin of effusions [ I ] . Of all tumor markers known to date, data are mostly available on CEA, indicating that the concentration of this antigen in effusions could improve the diagnostic accuracy of the cytological examination [ 2 , 3 ] . MCA (mucin-like carcinoma-associated antigen) is a high molecular weight glycoprotein (about 350 kD) which is also expressed in the majority of other adencarcinomas and in normal epithelial cells when detected by immunohistochemical methods[ I ] . Mucin-associated carbohydrate and peptide antigens are currently being investigated for their role in cancer diagnosis, monitoring for progression or metastases, immunotherapy and immunosupression 14-61, With respect to the premise that the new tumor marker MCA could be used as a diagnostic tool to detect metastasis-caused effusions, we determined the MCA concentrations in comparison wth the other carbohydrate antigens CA 19.9 and CA 125, as well as the well known tumor marker total sialic acid (TSA). in pleural effusions and in sera in lung carcinomas This study was undertaken in the Department of Biochemistrq., at Dokuz Eyliil University Medical Faculty, be twen September 1993 and February 1995: a total of 48 effusions were collected from 48 patients, who were subjected either to simple needle aspiration or to tube drainage for the diagnosis of a pleural effusion. Serum samples were obtained concurrently from all of these patients. All cytological examinations were carried out in the same sample as the tumor marker determinations. Classification of the groups were effectuated as follows ( I ) Malignant groups: 14 of those cases were suffering from histologically diagnosed lung malignancies (8 adenocarcinomas, 3 squamous cell carcinomas. 2 small cell carcinomas, and 1 malignant mesothelioma); (2) Benign group: 34 patients with various non-neoplastic disorders were included in this group (23 tuberculosis pleurisy, 5 pleuritis, 3 congestive heart disease, 2 pneumonia+cirrhosis, and I emppyema). The cut-off level between negative and positive ralues of each tumor marker was defined as the mean+2SD (% 95 percentile). Pleural effisions and uncoagulated blood samples were centrifuged at 2000 rpm for 10 minutes to remove cellular components. The supernatants were stored at -70 C until the performance of the tumor marker assays. The tumor markers MCA, CA 19.9 and CA 125 levels were measured in all samples by employing an enzyme-immunoassay method (EIA) (Hoffmann-La Roche, Basel, Switzerland). All measurements were done by the EIA photometer. automatically. The coefficients of lariation of the assays were determined to be as follows: the intra-assay coefficient of variation (CV) were 2.5 % for MCA, 3.4 %for CA 19.9, and 5.6 %for CA 125; the between-assay coefficients of variations ranged between 3.5 %and 9.6 % for MCA, 4.2 % and 11.5 % for CA 19.9, and 3.8 'Yo and 5.5 for CA 125. depending on the concentration of the control sera. TSA concentrations were determined by a modification of Ehrlich method. The reproducibility was checked at various sialic acid concentrations, and CV's were found to be <3.0 Yo in the whole measuring range. The lowest limit of detection defined as the concentration of N-acetylneuraminic acid that could be distinguished from blank with 95 YO confidence, was I mg/dl. The obtained tumor marker concentrations were statistically analyzed using the Student's-t test,Wilcoxon test, and Mann & Whitney test, accepting a p value below 5 Yo (p< 0.05) as significant. The results obtained for each group and for each tumor marker are presented in Table 1 Table I . MCA. CA 19.9. CA 125, and TSA in effusions and sera in lung cancer.

Implication of altered levels of plasma alpha(1)-acid glycoprotein and its derived sialic acid on plasma protein binding of trimethoprim in pigs in physiological and pathological states.

In growing pigs (0 to 158 days after birth), pregnant sows (0 to 90 days), and in pigs suffering from respiratory disease (Actinobacillus pleuropneumoniae), the plasma levels of alpha(1)-acid glycoprotein (AGP) and its derived sialic acid were determined, as was the meaning of their altered levels on the plasma protein binding of trimethoprim. The AGP level was very high immediately after birth (more than 10,000 mu g/ml), decreased markedly during 2 weeks after birth (about 700 mu g/ml) and thereafter stayed at a constant level (about 400 mu g/ml). Pregnant sows had a low AGP level with a narrow variation throughout pregnancy (about 190 to 260 mu g/ml). Pigs infected with A. pleuropneumoniae showed an increased AGP level (mean value; 732 mu g/ml) with a wide variation (range: 170-1,840 mu g/ml). N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) were sialic acid subtypes detected in porcine plasma. In growing pigs, the time course of changes in NANA concentrations was consistent with that of AGP, whereas that of NGNA was different, implying that NANA is a sialic acid subtype derived from porcine AGP, in contrast to NGNA. The relationship between AGP and NANA levels in growing pigs could be expressed by the following equation: NANA=0.14 x AGP + 159 mu g/ml, whereas that in pigs with respiratory disease could be expressed by NANA=0.O67 x AGP + 357 mu g/ml, indicating a low fraction of NANA in AGP in diseased pigs. The regression lines between the AGP level and the plasma protein binding of trimethoprim < or = 2,000 mu g of AGP/ml were similar as follows: binding(%)=0.O23 x GP + 34 in growing pigs and binding(%)=0.O22 x GP+29 in diseased pigs, implying a minor role of sialic acid residues in the binding of basic drugs to AGP. In conclusion, the wide change in plasma AGP levels in diseased pigs as well as during the initial growth phase can alter the plasma protein binding of basic drugs such as trimethoprim, probably leading to a change in drug disposition. The low sialylation of AGP in diseased pigs may not have a great influence on the binding of basic drugs to AGP, implying the quantitative importance of AGP.

Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms

A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of125I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of125I-LFA is not inhibited by 106-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 μl of serum were sufficient for the detection of major gangliosides.

Oral and Intraperitoneal Administration of N-Acetylneuraminic Acid: Effect on Rat Cerebral and Cerebellar N-Acetylneuraminic Acid

Rat pups were administered N-acetylneuraminic acid (NANA) by i.p. injection or via a feeding catheter for eight consecutive days beginning on d 14 of life. All pups were given the identical dose: 1.0 mg on d 1 and 2 and 1.2 mg on the remaining days, or approximately 20 mg/kg body weight per day. A control group was injected i.p. with glucose instead of NANA. On the morning of d 25, pups were decapitated, and the heads were frozen immediately in liquid nitrogen. The brains were later dissected and analyzed for both cerebral and cerebellar ganglioside and glycoprotein NANA. Administration of NANA by both oral and i.p. routes resulted in significantly more cerebral and cerebellar ganglioside and glycoprotein NANA than did glucose injection (with the exception of cerebellar glycoprotein NANA after NANA intubation). There were no significant differences in NANA concentration in these brain fractions for the two routes of NANA administration.

N-Acetylneuraminic acid concentrations in human milk oligosaccharides and glycoproteins during lactation

Human milk contains a variety of N-acetylneuraminic acid (NANA)-containing oligosaccharides, but the expected range of intake of sialic acid in this form by infants fed human milk is unknown. Two quite different amounts have been reported: 120 mg/liter in pooled, mature human milk (1) and 1400 mg/liter in the milk of a single woman on the 1st day of lactation (2). The normal range of NANA intake in human milk glycoproteins likewise does not appear to have been analyzed previously. Data presented here indicate that both human milk oligosaccharide and glycoprotein NANA decline exponentially over the first 2 months of lactation, decreasing little thereafter. During the first 2 months of lactation, milk from women delivering at term cannot be distinguished from that of women delivering significantly before term (less than 32 wks gestation) with regard to oligosaccharide and glycoprotein NANA. The parallel decrease of sialic acid in these fractions suggests a relationship between sialydation of human milk oligosaccharides and glycoproteins. Human milk NANA concentrations are discussed with regard to reports that exogenous administration of NANA can increase cerebral and cerebellar concentrations of NANA in glycoproteins and gangliosides, and produce long term changes in behavior in rats.

Use and limitations of serum total and lipid-bound sialic acid concentrations as markers for colorectal cancer.

Concentrations of total serum N-acetyl-neuraminic acid (NANA) by high-performance liquid chromatography (HPLC) and lipid-bound sialic acid (LSA) by resorcinol procedure were evaluated and compared to carcinoembryonic antigen (CEA) as markers for colorectal carcinoma. Elevated concentrations of NANA were found in 32% of patients with nonmalignant disorders, 28% of patients with localized cancer, and 87% of patients with metastatic cancer. All three markers correlated with the extent of metastasis of colorectal carcinoma. Strong correlation was found between NANA and LSA measurements, whereas measurement of the sialic acid markers provide information that can not be derived from the measurement of CEA. NANA and LSA show promise as supplemental markers for staging and monitoring colorectal cancer, but they are neither sensitive nor specific enough for cancer screening.

Plasma concentrations of N-acetylneuraminic acid in severe malnutrition.

In rat studies, circulating concentrations of N-acetylneuraminic acid (NANA) have been shown to be an indicator of NANA concentrations in the brain and functional brain activity, in relation to nutritional state and stimulation. Abnormal behaviour can be improved with exogenous NANA. In the present study, the plasma NANA concentration has been measured in children with severe malnutrition and compared with that in controls. NANA was measured colorimetrically in the plasma of twenty-three severely malnourished children (mean age 11.43 (SD 6.05) months) before and after recovery, and in thirty-four controls (mean age 14.28 (SD 7.32)months). In thirteen of the malnourished children, NANA was measured after infections had been treated with a course of antibiotics. Mean plasma NANA concentration was significantly higher in protein-energy malnutrition (PEM) (2.89 (SD 0.58) mumol/ml; n 23) compared with controls (2.13 (SD 0.37) mumol/ml; n 34, P less than 0.001). The levels remained high in PEM after infections had been treated (2.87 (SD 0.43) mumol/ml, n 13) but returned to control levels at recovery from PEM (2.14 (SD 0.24) mumol/ml). In contrast to the findings in rats, in malnourished children plasma NANA concentrations were not reduced and did not relate directly to nutritional state or, by inference, brain function. These findings do not provide any support for the use of exogenous NANA supplements to improve brain function in humans.

Salivary Secretion in Reflux Esophagitis

It has been speculated that impaired salivary flow and contents contribute to abnormal acid clearance in patients with reflux esophagitis. To test this hypothesis, salivary secretion of volume, bicarbonate, and N-acetylneuraminic acid was measured in patients with reflux esophagitis, in age- and sex-matched controls, and in healthy young volunteers. Resting saliva was collected by permitting the saliva to drip into a flask every 30 s. Parotid saliva was collected by means of a Lashley capsule during stimulation by oral infusion of 1 ml/min of 2% citric acid. Total mixed saliva was collected by a dental sucker during esophageal perfusion with water and 10-mM hydrochloric acid. The volume of resting saliva and its concentration of bicarbonate were similar in the three groups. The concentration and the output of N-acetylneuraminic acid were higher in the young volunteers than in the two other groups. The volume flow and the composition of stimulated parotid saliva were similar in the three groups of subjects. Volume of total mixed saliva and concentration of bicarbonate and N-acetylneuraminic acid during esophageal perfusion with water were higher in patients with reflux esophagitis than in age- and sex-matched controls and were similar to the values found in young healthy volunteers. Acid perfusion increased the secreted volume of total mixed saliva by 150% and the concentration of bicarbonate by 30% in healthy young volunteers, but not in patients with reflux esophagitis and in the healthy age- and sex-matched controls; the concentration of N-acetylneuraminic acid was left unaffected by acid perfusion in all three groups. These findings disprove the hypothesis of impaired salivation in reflux esophagitis. The age-related loss of salivary response to acidic esophageal perfusion might be the reason why reflux esophagitis with peptic lesions affects predominantly elder people.