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Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome-linked muscular dystrophy (mdx) model for DMD. Cmah -/- mdx mice can be induced to develop anti-Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah -/- mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: High consumption of red and processed meat is commonly associated with increased cancer risk, particularly colorectal cancer. Antibodies against the red meat-derived carbohydrate N-glycolylneuraminic acid (Neu5Gc) exacerbate cancer in "human-like" mice. Human anti-Neu5Gc IgG and red meat are both independently proposed to increase cancer risk, yet how diet affects these antibodies is largely unknown., Methods: We used world global data to demonstrate that colorectal cancer incidence and mortality are associated with increased national meat consumption. In a well-defined large cohort, we used glycomics to measure daily Neu5Gc intake from red meat and dairy, and investigated serum as well as affinity-purified anti-Neu5Gc antibodies. Based on 24-h dietary records, daily Neu5Gc intake was calculated for 19,621 subjects aged ≥ 18 years of the NutriNet-Santé study. Serum and affinity-purified anti-Neu5Gc antibodies were evaluated by ELISA and glycan microarrays in representative 120 individuals, each with at least eighteen 24-h dietary records (aged 45-60, Q1-Q4; aged > 60, Q1 and Q4; 10 men/women per quartile)., Results: We found that high-Neu5Gc diet, gender, and age affect the specificity, levels, and repertoires of anti-Neu5Gc IgG immune responses, but not their affinity. Men consumed more Neu5Gc than women, mostly from red meat (p = 0.0015), and exhibited higher overall serum anti-Neu5Gc IgG levels by ELISA (3.94 ng/μl versus 2.22 ng/μl, respectively; p = 0.039). Detailed glycan microarray analysis against 56 different glycans revealed high Neu5Gc-specificity with increased anti-Neu5Gc IgG and altered repertoires, associated with higher consumption of Neu5Gc from red meat and cow dairy. Affinity purification of serum anti-Neu5Gc antibodies revealed increased levels and biased array repertoire patterns, without an increase in antibody affinity, in individuals consuming higher Neu5Gc levels. Furthermore, in a high-meat diet, antibody diversity patterns on glycan microarrays shifted towards Neu5Gcα3-linked glycans, increasing the α3/α6-glycans ratio score., Conclusions: We found a clear link between the levels and repertoire of serum anti-Neu5Gc IgG and Neu5Gc intake from red meat and dairy. These precise rational methodologies allowed to develop a Gcemic index to simplify the assessment of Neu5Gc in foods that could potentially be adapted for dietary recommendations to reduce cancer risk.

Strong discrepancies in published data on the levels and epitope specificities of antibodies against the xenogenic N-glycolyl forms of sialoglycans (Hanganutziu-Deicher Neu5Gcɑ2-3Galβ1-4Glc and related antigens) in healthy donors prompted us to carry out a systematic study in this area using the printed glycan array and other methods. This article summarizes and discusses our published and previously unpublished data, as well as publicly available data from the Consortium for Functional Glycomics. As a result, we conclude that (1) the level of antibodies referred to as anti-Neu5Gc in healthy individuals is low; (2) there are antibodies that seem to interact with Neu5Gc-containing epitopes, but in fact they recognize internal fragments of Neu5Gc-containing glycans (without sialic acids), which served as antigens in the assays used and; (3) a population capable of interacting specifically with Neu5Gc (it does not bind the corresponding NAc analogs) does exist, but it binds the monosaccharide Neu5Gc better than the entire glycans containing it. In other words, in healthy donors, there are populations of antibodies capable of binding the Neu5Gc monosaccharide or the inner core -Galβ1-4Glc, but very few true anti-Neu5Gcɑ2-3Galβ1-4Glc antibodies, i.e., antibodies capable of specifically recognizing the entire trisaccharide., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

N-glycolylneuraminic acid (Neu5Gc)-containing glycans are a prominent form of aberrant glycosylation found in human tumor cells and have been proposed as cancer biomarkers. The B subunit of the subtilase cytotoxin (SubB) produced by Shiga toxigenic Escherichia coli recognises Neu5Gc containing glycans. We have previously engineered this lectin, SubB2M, for greater specificity and enhanced recognition of Neu5Gc-containing glycans. Here we further explore the utility of SubB2M to detect Neu5Gc tumor biomarkers in sera from patients with ovarian cancer. Using surface plasmon resonance (SPR) we show that SubB2M can detect the established ovarian cancer biomarker, CA125, in a highly sensitive and specific fashion in the context of human serum. These studies established conditions for screening serum samples from patients with ovarian cancer for Neu5Gc glycans. We found that serum from patients with all stages of ovarian cancer had significantly elevated mean levels of Neu5Gc glycans compared to normal controls. Serum from patients with late stage disease (stages IIIC, IV) had uniformly elevated levels of Neu5Gc glycans. Detection of Neu5Gc-glycans using SubB2M has the potential to be used as a diagnostic ovarian cancer biomarker, as well as a tool for monitoring treatment and disease progression in late stage disease., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

N-acetylneuraminic acid (Neu5Ac) and N-acetylglycolylneuraminic acid (Neu5Gc), two acylated derivatives of 9-C carboxylated monosaccharides, are involved in a number of biological processes as modulators of glycoconjugates. A partially automated method is here presented for determination of these sialic acids in the two most important biofluids for clinical analysis: serum and urine. For this purpose, a solid-phase extraction (SPE) workstation was on-line connected to an LC-MS/MS triple quadrupole mass detector. Hydrolysis to release sialic acids bound to glycoconjugates and derivatization were the two steps implemented as sample preparation prior to SPE-LC-MS/MS analysis. Following thorough optimization of the SPE and LC-MS/MS conditions, the analytical method was validated using the standard addition approach to assess the presence of matrix effects. The proposed method affords detection limits of 0.03ng/mL and 0.04ng/mL for Neu5Ac and Neu5Gc, respectively. The precision (expressed as relative standard deviation) was 1.7 and 4.6% for within-day variability, and 4.8 and 7.2% for between-days variability. Accuracy, estimated using spiked (between 1 and 50ng/mL) and non-spiked samples of both biofluids, ranged from 95.2 to 99.6%. The method was applied to human serum and urine of healthy volunteers, thus showing its suitability for application in both clinical and research laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Bovine red blood cells (RBCs) do not exhibit any aggregation tendency in autologous plasma and, therefore, all bovine rouleaux obtained in vitro are regarded as artificial. The present study reports the bovine RBC rouleau formation by either bovine or human fibrinogen and Ca2+ ions. The phenomenon was induced through two-step cell incubation: in 0.9% NaCl and 1% bovine albumin at 37 degrees C for 30 min followed by 20 hrs incubation at 30 degrees C in the fresh solution supplemented with 10 mM glucose. Its mechanism is unknown. During the incubation the number of N-glycolylneuraminic acid molecules per cell decreased from 48.1 to 44.9 amoles, which accounted for 7%. The treatment of RBCs with V. cholerae sialidase under the same conditions resulted in a 94% drop in the Neu5Gc quantity and did not induce the rouleau formation in the same fibrinogen preparation. The preliminary results rise a question whether the bulk of sialic acid is required in the aggregation of bovine erythrocytes under static conditions. Only a minor pool of Neu5Gc seems to be responsible for suppression of the phenomenon., (Copyright 2004 IOS Press)

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) was developed by using high-performance capillary electrophoresis (HPCE) with UV detection at 195 nm. NANA and NGNA were separated directly and analyzed without pre- or postcolumn derivation. The detection limit of NANA is 9.6 x 10(-6) mol L(-1) and for mass 3.879 x 10(-14) mol (39 fmol). This method was applied for the determination of NANA in 30 normal human and 72 cancer patients. The results demonstrated that NANA in the sera of cancer patients increased significantly as compared with the normal human (P < 0.001). The new method is simple and sensitive, and is suitable for basic research and clinical application to malignant tumors.

The objectives of this study were the quantification of the two major sialic acid (Sia) forms - N-acetylneuraminic (Neu5Ac) and N-glycolylneuraminic acids (Neu5Gc) - in serum before and after surgical treatment of early endometrial cancer and the relation of their levels with the progress of surgical therapy. The major Sia forms were liberated from sera glycoconjugates by mild acid hydrolysis, separated as per-O-benzoylated derivatives by a highly sensitive reversed-phase HPLC method and detected at 231 nm. Total Sia content in sera of healthy women was not related to age and body weight. Neu5Ac was identified as the major Sia in sera from both cancer patients, healthy individuals as well as in tissue specimens (> or = 94% of total Sia). In patients with endometrial cancer the total Sia level before surgical treatment (709.5 +/- 306.5 mg/l) was significantly higher (p < or = 0.0001) than that of the control group (213.5 +/- 88.7 mg/l). The elevation in Sia level was exclusively due to Neu5Ac. Following surgical therapy, serum Neu5Ac levels (699.4 +/- 305.6 mg/l) were significantly decreased (305.9 +/- 114.5 mg/l). In one case, where Neu5Ac level was increased 15 days and eight months after surgery (1.8 and 2.5 times as compared to control, respectively), a metastasis not detected during surgery was recorded. The obtained results suggest that Neu5Ac level in serum may be used as a tumor marker in evaluating the suitability of surgical treatment in early endometrial cancer.

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.

Serum and tissue specimens from healthy C57BI mice and from mice with Lewis' lung cancer after metastasis were analyzed for N-acetyl- and N-glycolylneuraminic acid by high-performance liquid chromatography. Both neuraminic acids were present, while N-glycolylneuraminic acid was the predominant sialic acid in all tissues. Samples from mice with metastatic cancer showed a significant increase (67-200%) of total sialic acids mainly as a result of increased N-glycolylneuraminic acid synthesis. These results suggest that cancer metastasis in various tissues is closely associated with increased synthesis of the predominant neuraminic acid and may help to understand the underlying mechanisms of tumor development.

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.

Porcine transmissible gastroenteritis virus (TGEV) has been shown to agglutinate erythrocytes using alpha 2,3-linked sialic acid on the cell surface as binding site. The hemagglutinating activity requires the pretreatment of virus with neuraminidase. We obtained evidence that TGEV recognizes not only N-acetylneuraminic acid but also N-glycoloylneuraminic acid, a sialic acid present on many porcine cells.

A simple, rapid, and extremely sensitive method for determining N-acetyl- and N-glycolylneuraminic acids in serum and in submandibular, sublingual, and parotid glands using high-performance liquid chromatography with a fluorometric detector is described. The neuraminic acids contained in the samples are released in the presence of 2 M CH3COOH by means of microwave hydrolysis (only 10 min required) and are subsequently converted using 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-ketoacids, into highly fluorescent derivatives. In order to optimize the release of sialic acids and to minimize the effect of destruction, the following analytical variables were investigated: temperature, time, and concentration of the acid. Within 12 min after derivatization, the compounds were separated on a reversed-phase column by means of isocratic elution using a mobile phase of water (pH 3 with H3PO4)-methanol-acetonitrile (86:6:8, v/v). Fluorescence detection was performed at an excitation wavelength of 373 nm and an emission wavelength of 448 nm.

N-Acetylneuraminic acid (Neu5Ac) and N-glycoloylneuraminic acid (Neu5Gc) are distributed widely in nature. Using a Carbopac PA-1 anion exchange column, we have determined the ratios of Neu5Ac and Neu5Gc in hydrolysates of platelets and their precursors: a rat promegakaryoblastic (RPM) cell line and a human megakaryoblastic leukemia cell line (MEG-01). The ratio of Neu5Gc:Neu5Ac in cultured RPM cells is 16:1, whereas in platelet rich plasma and cultured MEG-01 cells it is 1:38 and 1:28, respectively. The nature of these sialic acids from RPM cells was verified using thin layer chromatography and liquid secondary ion mass spectrometry. The relevance of increased Neu5Gc levels in early stages of development is discussed.

Blood group incompatibility causes transfusion reactions and neonatal isoerythrolysis in cats. We investigated the molecular nature of the blood group antigens from cats that had blood type A, B, and AB erythrocytes. Naturally occurring anti-type B antibodies, Triticum vulgaris lectin, monoclonal antibody (MoAb) 32-27, and MoAb R-24 were used in agglutination tests, Western blots, and thin-layer chromatography (TLC) enzyme immunostaining. Type A erythrocytes had NeuGc-NeuGc-Galactose-Glucose-Ceramide ([NeuGc]2GD3) where NeuGc represents N-glycolylneuraminic acid, and NeuAc-NeuGc-GD3, where NeuAc represents N-acetylneuraminic acid, and may have [NeuGc]2 disialylparagloboside and NeuAc-NeuGc-disialylparagloboside. Type B erythrocytes only had [NeuAc]2GD3. Type AB erythrocytes had [NeuGc]2GD3, NeuAc-NeuGc-GD3, and [NeuAc]2GD3. Blood group antigens were also found on a 50-Kd membrane protein. We conclude that type B erythrocytes are characterized by [NeuAc]2GD3 as the only form of this ganglioside and the presence of NeuAc on a 50-Kd membrane protein. NeuGc is the major determinant of the A antigen; specifically, [NeuGc]2GD3 is the major glycolipid form. The A antigen is also present on a 50-Kd membrane protein.

The aim of this study was to produce large liver tumors reliably, and to diagnose the tumors during development. Therefore, New Zealand white rabbits were treated with N-nitrosodiethylamine orally three times per week by gavage and were examined by clinical-chemical assay at regular intervals during the average treatment period of 14 months. The total cumulative dose was 1200 mg N-nitrosodiethylamine over 14 months. After a short treatment period the initial dose of 3 mg/kg had to be reduced to 1.5 mg/kg. In all 11 treated animals (100%) liver tumors were seen at the end of the study. Four control animals did not show any neoplastic changes. Clinical parameters investigated were for an assessment of liver function, total protein, urea, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, albumin and neuraminic acid as well as some serum electrolytes. The in vivo diagnosis of liver tumors based on changes in these parameters proved to be relatively unreliable. The liver enzyme tests and urea concentration only yielded significant changes when the liver tumors were very large. Changes in neuraminic acid levels were the most reliable indicator for the presence of a liver tumor in this animal model. In the 11 treated animals, serum values of this marker increased towards the end of the study by an average of 300 mg/dl. The induced tumors were mainly hepatocellular carcinomas. Only in 1 animal was a hepatocellular adenoma found. Further primary tumors diagnosed were six adenomas in the kidneys and two uterus adenomas, as well as nasal cavity tumors (two papillomas, one carcinoma, one adenoma and one adenocarcinoma). In 70% of the treated rabbits the hepatocellular carcinomas had metastasized to the lungs.

Genetic polymorphism was observed in the sialic acid species constituting the terminal sugar residues of hematosides from dog erythrocytes. One was N-acetylneuraminic acid and the other phenotype was N-glycolylneuraminic acid, regulated by an autosomal dominant allele (Yasue, S., Handa, S., Miyagawa, S., Inoue, J., Hasegawa, A., & Yamakawa, T. (1978) J. Biochem. 83, 1101-1107). In this study we analyzed blood samples from 1,591 dogs of 36 breeds and demonstrated that the expression of N-glycolylneuraminic acid was limited to several breeds of oriental dogs in spite of its dominant nature. Moreover, the incidence of N-glycolylneuraminic acid was higher in native breeds of northern China, Korea and the southern part of Japan than in other oriental breeds. On the other hand, the Hokkaido-dog is unique in not expressing N-glycolylneuraminic acid. These results suggest that the native breeds in the southern part of Japan came from northern China via the Korean peninsula in contrast with indigenous breeds of the northern part of Japan.

The authors determined the level of neuraminic acid in the serum of 50 patients with encephalomalacia and 25 patients with cerebral haemorrhages on the 1st, 3d and 7th days of the disease. Twenty healthy subjects served as controls. A statistically significant rise in the concentration of this substance was observed in both groups of patients throughout the whole period of observations.

A technique described by Cham and Knowles allows the extraction of plasma phospholipides, triglycerides, cholesterol and free fatty acids without denaturing the proteins. The method has been slightly modified by us. Under our conditions, the yield of lipid extraction is comparable to the method of Folch. It has the advantage of not extracting mineral phosphorous and blood glucose. Thus gangliosides can be also easily determined. We confirmed by immunoelectrophoresis that the antigenic properties of plasmatic proteins remain after lipid extraction.

Hematoside from dog erythrocyte membrane was previously considered to contain a mixture of N-acetyl- and N-glycolyl- neuraminic acids. However, the hematoside preparation used in the previous study was obtained from pooled blood of several dogs, and individual variation in hematoside was not examined. In this work, hematosides of erythrocytes from 31 mongrel dogs and 108 dogs of 23 breeds were examined individually by thin-layer chromatography, and the component sialic acids were analysed by gas-liquid chromatography. Individual dogs had either NAN-hematoside or NGN-hematoside: dogs with N-glycolyl-neuraminic acid also had a trace of N-acetyl-neuraminic acid, but dogs with N-acetyl-neuraminic acid had no detectable N-glycolyl-neuraminic acid. A few mongrel dogs, some Kai dogs, Kishu dogs, Japanese spaniels and most Shiba dogs had NGN-hematoside, whereas all European dogs had NAN-hematoside and no NGN-hematoside. From pedigrees of some families, inheritance of NGN-hematoside was found to be autosomal dominant. NGN-hematoside is possibly one of dog blood group substances. The sialic acid of delipidized ghost protein of dogs with NGN-hematoside was N-glycolyl-neuraminic acid, and that of dogs with NAN-hematoside was N-acetyl-neuraminic acid. The sialic acid of plasma protein was mainly N-acetyl-neuraminic acid in all dogs.

Changes in different protein fractions during immobilization stress were demonstrated. The concentration of albumins increased and that of all globulin fractions decreased, especially the concentration of alpha 2, and gamma globulins. Increased concentration of the carbohydrate components of serum proteins was also observed. Since the latter change was associated with a fall in globulin concentration, and globulins, contrary to albumins, contain carbohydrate components, it may be supposed that the proteins investigated during stress have molecules rich in carbohydrates.

The neuraminic acid level in the serum of 588 patients was determined. The patients suffered from bronchial carcinomas other malignant diseases (mastocarcinoma, carcinoma of the cervix and the body of the uterus, Hodgkin's lymphoma, non-Hodgkin's lymphoma), and benign pulmonary diseases (tuberculosis of the lungs and chronic obstructive pulmonary diseases). The average level of 162 patients with bronchial carcinomas is 3,449 mumol/ml of serum; thus it is significantly higher than the average level of 98 sound control persons (2,336 mumol/ml) and 88 patients with benign pulmonary diseases (2,733) mumol/ml). The other patients with malignant diseases also presented an increased concentration of neuraminic acid in the serum, however, this increase was not as significant as for the patients with bronchial carcinomas. The possible role of neuraminic acid as biological "marker" is discussed.

The serum concentration of neuraminic acid and the carcino-embryonic antigen (CEA) were measured in 178 patients with diagnosed mammary carcinoma and 114 control patients. Among these 178 patients, 16 showed a local recurrence, 68 were tumor-free, and 94 had distant metastases. Only 42 out of these 94 patients kept the check-up appointments. During six months, they were regularly examined and treated with cytostatic drugs. The neuraminic acid levels corresponded mostly to the clinical state and the response to chemotherapy of these patients. All of the 94 patients with generalized mammary carcinoma had a significantly increased neuraminic acid concentration in the serum (p less than 0.001). The serum levels were not modified in 16 patients with local recurrences and in 68 tumor-free patients. The CEA values did not correspond to the serum neuraminic acid levels and the clinical state of the patients. The increase of the neuraminic acid concentration in the serum of these 42 patients with metastatic mammary carcinomas show that this parameter is a valuable indicator for the progression of this disease.

10-20ml erythrocytes of 17 healthy subjects were separated from leukocytes by passage through cotton. The erythrocytes were haemolyzed in hypotonic buffer and the ghosts isolated by centifugation. Lipid extraction was performed and the gangliosides purified by molselect chromatography. Fractionation of gangliosides was carried out by thin layer chromatography. The quantitative and qualitative determination was performed by thin layer scanning with a High Speed TLC-Scanner Shimadzu CS 920. The sensitivity of the method was about 100 ng lipid bound neuraminic acids. The concentration of neuraminic acids in the total gangliosides was about 14 microgram/20 ml erythrocytes. The distribution of the 4 different ganglioside fractions which could be detected after staining with Svennerholm reagent were: GM3=16%, GM1=52%, GD1a=22%, GT1=90%.

Lectins from Canavalia ensiformis, Phaseolus vulgaris, and Triticum vulgare react with arylamidase, alkaline phosphatase, gamma-glutamyltransferase, and cholinesterase of human sera by formation of enzymatically active, mostly insoluble complexes. Arylamidase, alkaline phosphatase, and cholinesterase react more intensely in sera of healthy people than in sera of patients with liver and neoplastic diseases. Arylesterase is bound to a distinct degree only by concanavalin A. The enzymes mentioned above also react slightly with the following lectins in order of decreasing intensity: Ricinus communis, Arachis hypogaea, Helix pomatia, Glycine max, Dolichos biflorus, and Ulex europaeus. Though multiple forms containing less sialic acid are favourably bound, preincubation with neuraminidase does not improve the reaction except with soybean lectin. Since higher concentrations of lectins react also with fast moving fractions of high sialic acid content, no steric hindrance of the binding between lectins and sialoenzymes is supposed, as concluded from determination of the total enzyme activity.

It is often impossible to make a clinical distinction between acquired and hereditary acute angioneurotic oedema. Investigation of the complement system is indispensable for this diagnosis to be established. The value of total complement and C4 and C2 are lowered in the sera in the hereditary form (44 cases) and normal in the acquired type (68 cases). The use of tests for the activation of C1 esterase "in vitro" is useful to distinguish these two types of oedema as has been demonstrated by the formal measurement of C1 esterase inhibitor.