Creatinine concentration in plasma from dog, rat, and mouse: a comparison of 3 different methods.

BACKGROUND: There are numerous methods for analyzing creatinine concentration in plasma, including the Jaffé alkaline picrate method in various modifications, enzymatic tests, and chromatographic methods. OBJECTIVE: The purpose of this study was to evaluate whether an enzymatic method could replace a Jaffé method for routine creatinine measurements in plasma from dogs, rats, and mice. The enzymatic method and a compensated Jaffé method were tested against a high-pressure liquid chromatography (HPLC) method, regarded as the gold standard for creatinine measurement. METHODS: Heparinized plasma samples were obtained from 20 beagle dogs, 20 Wistar rats, and 20 CD1-strain mice. The 2 test kits (Roche Diagnostics), Creatinine Jaffé Compensated and the enzymatic Creatinine Plus Version 2 reagent, were used on a Cobas Integra 400. The Jaffé compensated method used a calibration adjustment of 18 micromol/L to correct for the protein matrix in serum and plasma. The HPLC method was an isocratic method using a weak cation-exchange column following protein precipitation. RESULTS: Creatinine concentrations obtained using the enzymatic and the Jaffé methods differed significantly from the results obtained by the HPLC method. For dog plasma, mean values of 61.2, 61.8, and 67.8 micromol/L were obtained by the compensated Jaffé, enzymatic and HPLC methods, respectively. In the rat, respective mean values were 26.7, 21.9, and 23.0 micromol/L, and in the mouse, respective mean values were 14.2, 5.4, and 9.2 micromol/L. CONCLUSION: The enzymatic method can replace the Jaffé method for plasma creatinine determination in dogs, rats, and mice because results from the enzymatic method were closer to HPLC values than were those of the Jaffé method.

Identification of a missense mutation (G329A;Arg(110)--> GLN) in the human FUT7 gene.

The human FUT7 gene codes for the alpha1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe(x)) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe(x) and SLe(x)-related epitopes. One individual showed lower levels of SLe(x) expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg(110) --> Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg(110) is conserved in all previously cloned vertebrate alpha 1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of < or =1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe(x) and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjögren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

A lectin immunosensor technique for determination of alpha(1)-acid glycoprotein fucosylation.

The fucosylation of alpha(1)-acid glycoprotein (AGP), an acute-phase protein, is known to change in association with inflammatory diseases. Thus, fucosylation of AGP could be a potential diagnostic or prognostic marker. The change in fucosylation has previously been investigated using crossed affinoimmunoelectrophoresis, high-pH anion-exchange chromatography, and lectin ELISA. This study describes a surface plasmon resonance-based affinity biosensor assay for quantification of the fucosylation of AGP. Diluted EDTA plasma or serum was injected directly in a BIACORE 2000 biosensor. AGP was captured on the sensor surface using immobilized antibodies and a fucose-binding lectin from Aleuria aurentia was then used for the detection of fucosylation. The feature of the biosensor makes it possible to determine both the amount of bound AGP and the amount of bound lectin. Using a calibration curve it was possible to obtain a fucosylation ratio that was independent of AGP concentration. The assay was validated against a lectin ELISA and used to follow inflammation in patients with severe burns.

Changes in glycosylation of human bile-salt-stimulated lipase during lactation.

Bile-salt-stimulated lipase (BSSL) is an enzyme in human milk, which is important for the fat digestion in the newborn infant. BSSL is highly glycosylated and includes one site for N-glycosylation and several sites for O-glycosylation. BSSL has previously been found to express Lewis a, Lewis b, and Lewis x carbohydrate antigens. In this study, glycosylation of BSSL was studied at different times during lactation. BSSL was purified from milk collected individually from four donors at several different times during the first 6 months of lactation. The BSSL glycans were characterized through monosaccharide analysis, high-pH anion-exchange chromatography, matrix-assisted laser desorption-ionization mass spectrometry, and ELISA. Both total carbohydrate content and relative amount of sialic acid were higher in BSSL from the first lactation month as compared to BSSL from milk collected later in lactation. BSSL from the first lactation month also showed a different composition of sialylated O-linked glycans and the N-linked oligosaccharides consisted of lower amounts of fucosylated structures compared to later in lactation. We also found a gradual increase in the expression of the carbohydrate epitope Lewis x on BSSL throughout the lactation period. This study shows that glycosylation of BSSL is dependent on blood group phenotype of the donor and changes substantially during the lactation period.

Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance.

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.

Serum sialic acid and sialoglycoproteins in asymptomatic carotid artery atherosclerosis. ARIC Investigators. Atherosclerosis Risk in Communities.

Serum total sialic acid (S-TSA) is a recently identified risk marker for atherosclerosis and cardiovascular mortality. The purpose of this study was to evaluate the influence of three sialic acid rich glycoproteins (orosomucoid, haptoglobin, and alpha1-antitrypsin) on the relationship between S-TSA and carotid atherosclerosis. The mean S-TSA was 0.045 g/l higher among cases than controls (P<0.001) in 310 45-64 year-old male and female pairs of carotid atherosclerosis cases and disease-free controls from the Atherosclerosis Risk in Communities (ARIC) Study. Also mean serum levels of the glycoproteins were significantly higher in cases compared to controls. In a conditional multiple logistic regression model with the glycoproteins as independent variables, orosomucoid was correlated most strongly with case control status. However, when incorporated into the mathematical model, S-TSA not only contributed additional information as to the risk of atherosclerosis; none of the three glycoproteins contributed further once S-TSA had been accounted for. Thus, some other source of serum sialic acid or variations in the degree of sialylation of glycoproteins may be essential for understanding the relation between S-TSA and atherosclerosis.

Staphylococcal enterotoxin-A-induced in-vitro adhesion of HL-60 cells to endothelial cells involves both selectin and integrin families of cell adhesion molecules.

In-vivo exposure to the bacterial superantigen Staphylococcal enterotoxin-A (SEA) induces an inflammatory response characterized by rapid extravasation of leucocytes and release of excessive amounts of cytokines. We have utilized an in-vitro adhesion assay to understand the molecular mechanisms responsible for SEA-induced extravasation of leucocytes. Stimulation of human umbilical cord endothelial cells (HUVEC) with increasing concentrations of recombinant SEA (rSEA) did not influence the in-vitro adhesion of HL-60 cells to HUVEC, whereas stimulation of HUVEC by interleukin (IL)-1beta supported adhesion of HL-60 cells. Increased adhesion of HL-60 cells to HUVEC was noted upon stimulation of endothelium with culture medium obtained from human peripheral blood mononuclear cells (PBM) stimulated with recombinant SEA for 24 (CM-SEA 24 h), 72 (CM-SEA 72 h) and 120 h (CM-SEA 120 h), but not after stimulation with culture medium obtained from control human peripheral blood mononuclear cells (CM), suggesting that soluble factors present in the supernatants play a major role in SEA-induced cell adhesion. While CM-SEA 24 and 72 h induced both a rapid (4 h) and delayed type of adhesion, CM-SEA 120 h only induced a delayed type of adhesion. Stimulation of PBM by SEA resulted in increased levels of IL-1beta, IL-2 and interferon (IFN)-gamma after 24h. Further stimulation for 72-120h resulted in a significant increase in the levels of IL-1beta, IFN-gamma and tumour necrosis factor (TNF). Stimulation of PBM with SEA also resulted in increased levels of soluble and L-selectin in the cell supernatants. Increased cell-surface expression of E-selectin, ICAM-1, HLA-DR and VCAM-1 was detected on HUVEC stimulated with CM-SEA media. While E-selectin and VCAM were induced on HUVEC within a few hours, induction of ICAM and HLA-DR required a longer induction period. Adhesion of HL-60 cells to HUVEC treated with CM-SEA was inhibited by monoclonal antibodies (MoAbs) against both the selectin and integrin families of cell adhesion molecules, suggesting that multiple pathways contribute to SEA-induced leucocyte extravasation. The results suggested that selectin-dependent adhesion was more prominent during the early phase while integrin-induced adhesion occurred at a later stage.

Temperature effects in high-performance anion-exchange chromatography of oligosaccharides.

High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection has been widely used for analysis of mono-, oligo- and polysaccharides. Many factors that affect separation of carbohydrates by HPAEC have been evaluated, however effect of temperature has not been carefully studied. In the present study, neutral and sialylated oligosaccharides from human milk and different types of N-linked oligosaccharides were analysed by HPAEC at temperatures ranging from 13 to 30 degrees C. N-Acetyl neuraminic acid, Galacturonic acid and stachyose were also analysed since they have been used as internal standards when analysing various oligosaccharides by HPAEC. All oligosaccharides showed decreased retention times with increased temperature. Even small differences (i.e. +/- 5 degrees) resulted in considerable changes in retention times. In addition, individual oligosaccharides showed relative changes in retention time with increased temperature. By changing the temperature, a switch in elution of order of individual oligosaccharides were sometimes found. These results show that retention times relative to an internal standard cannot be used for oligosaccharide identification unless temperature is carefully controlled. Regulation of temperature is also a valuable tool in achieving optimal separation of oligosaccharides by HPAEC.

Use of weak monoclonal antibodies for affinity chromatography.

When using weakly interacting ligands in affinity chromatography, it is possible to take advantage of a true chromatographic process in the separation, as compared with traditional affinity chromatography which is rather an on/off process. In this work, weak monoclonal antibodies were immobilized on a silica and a perfusion-type support (POROS AL) and used for high-performance liquid affinity chromatography (HPLAC). Similar carbohydrate antigens were separated under isocratic conditions according to their weak interaction with the immobilized monoclonal antibody. The affinity of the antibodies was adjusted with temperature and pH to modify the separation. The productivity of the chromatographic system was increased with the immobilized perfusion support but at the expense of loss of plate numbers. This study clearly demonstrates the potential of weak affinity biological interactions as a basis for chromatographic separation.

Detection of low-molecular-weight heparin oligosaccharides (Fragmin) using surface plasmon resonance.

During the last decades there has been a growing realization of the central biological role that oligosaccharides and oligosaccharide-protein interactions play. One of the most striking examples is the use of heparin and low-molecular-weight heparin oligosaccharides (Fragmin) to modify blood coagulation. Several monoclonal antibodies directed against glycosaminoglycan structures have been produced. However, their clinical use is limited by the difficulty of detection systems for oligosaccharides. In the present study we used a monoclonal antibody directed against heparin oligosaccharides prepared by partial nitrous acid deamination of heparin. Using a biosensor (BIAcore), purified antibody was immobilized on sensor surfaces and binding of oligosaccharide was measured by surface plasmon resonance. Using this technique, it was possible to quantitate low-molecular-weight heparin oligosaccharides in nanomolar concentrations.

Interferon-gamma enhancement of E-selectin expression on endothelial cells is inhibited by monensin.

The expression of E-selectin reaches a maximum 4-6 h after stimulation of human umbilical vein endothelial cells (HUVEC) in vitro with tumour necrosis factor-alpha (TNF-alpha) and then declines to basal level within 24 h. If interferon-gamma (IFN-gamma) is added to the cell culture medium together with TNF-alpha the surface expression of E-selectin is augmented and prolonged in a synergistic way. The aim of the present study was to investigate if altered protein glycosylation could explain the IFN-gamma induced persistent surface expression of E-selectin. SDS-PAGE analysis of HUVEC glycoproteins, metabolically radiolabelled in the carbohydrate portion, indicated that addition of IFN-gamma produced an altered protein glycosylation. Lectin blot analysis using the Sambucus nigra agglutinin lectin also indicated differences in protein glycosylation when HUVEC were incubated with IFN-gamma/TNF-alpha compared to TNF-alpha alone. The kinetics of surface expression of E-selectin were measured using a cell ELISA assay. When HUVEC were incubated with monesin, a potent inhibitor of late Golgi function, together with both TNF-alpha and IFN-gamma, the additive effect of IFN-gamma on E-selectin expression was almost abolished. Since monensin is known to affect glycosylation processing, this experiment suggested that the IFN-gamma induced change in protein glycosylation might induce the prolonged surface expression of E-selectin. However, when HUVEC were cultured with IFN-gamma/TNF-alpha in the presence of several different inhibitors of N-glycosylation processing, no significant effect of E-selectin expression was observed. Regulation of adhesion molecule expression after activation of endothelial cells is likely to play a pivotal role for the inflammatory response. Further studies are needed to understand the mechanisms underlying this regulation.

Glycosylation of bile-salt-stimulated lipase from human milk: comparison of native and recombinant forms.

Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19-26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types of N-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short type O-glycans with a high content of sialic acid. Interestingly, the estimated number of O-glycans attached to native BSSL was lower than that for the recombinant forms.

Glycosylation of alpha1-acid glycoprotein in inflammatory disease: analysis by high-pH anion-exchange chromatography and concanavalin A crossed affinity immunoelectrophoresis.

High-pH anion-exchange chromatography with pulsed amperometric detection is a highly sensitive technique that can be used for detecting changes in sialylation and fucosylation, as well as different branching patterns of N-linked oligosaccharides in glycoproteins. We examined the N-glycans of alpha1-acid glycoprotein obtained from twelve patients with various inflammatory conditions with this technique, as well as traditional concanavalin A crossed affinity immunoelectrophoresis. We found the chromatographic profiles of N-glycans in all patients with rheumatoid arthritis to be very similar, but significantly different from normal controls. N-glycans from patients with ulcerative colitis also showed specific alterations in their chromatographic profiles. However, some heterogeneity was found between these patients, perhaps reflecting changes in glycosylation secondary to certain states of the disease, or to medical treatment. We conclude that this technique is useful for detailed mapping of glycosylation changes in alpha1-acid glycoprotein in clinical samples, and that it may be used to further increase our knowledge about glycosylation changes in response to inflammatory disease.

Accurate and precise isotope dilution mass spectrometry method for determining glucose in whole blood.

An accurate and precise method to determine glucose concentration in whole blood is presented. The method, based on isotope dilution gas chromatography-mass spectrometry (ID GC-MS), was developed to be used as a Reference Method for determining glucose concentration in capillary or venous whole blood. Blood samples and standards are pipetted manually with "microcap" micropipettes, which makes it possible to collect samples even at the patient's bedside. Glucose is quantified as its aldononitrile pentaacetate. [13C6]Glucose is used as an internal standard. Assay of Seronorm and Pathonorm L and H controls by ID GC-MS gave within-run CVs of 0.66%, 0.96%, and 0.92%, respectively. For whole blood with glucose concentrations in the low, normal, and high ranges, the within-run CVs were 1.27%, 0.91%, and 0.78%, respectively. The between-run CV for glucose calculated from 36 separate single analyses of Seronorm was 1.44%. In an accuracy assessment test of the HemoCue blood glucose analyzer, 140 capillary blood samples were measured in parallel after split-sampling. For all samples the HemoCue analyzer results had a mean bias of +2.0% compared with the ID GC-MS results.

Serum sialic acid and its correlates in community samples from Akita, Japan and Minneapolis, USA.

OBJECTIVE: The concentration of serum total sialic acid (S-TSA) is one recently investigated risk marker for cardiovascular mortality and atherosclerosis. Since the mortality from coronary heart disease is higher in the United States than in Japan, one could expect the S-TSA to be higher among Caucasian US citizens than among Japanese citizens, a hypothesis that is tested in this study. DESIGN: Cross-sectional study of population-based samples of Japanese and US Caucasian men and women. SETTING: The rural community Akita, Japan, and the suburbs of Minneapolis, Minnesota. SUBJECTS: These were 75 consecutive men and women from Akita and Minneapolis respectively aged 47-69 years in 1990. People who had smoked cigarettes during the past 5 years; who had a history of diabetes mellitus, liver disease, coronary heart disease, or stroke; or who were taking anticoagulants were excluded. OUTCOME MEASURES: Serum total sialic acid levels in male and female Japanese and US Caucasian subjects with adjustment for age, systolic blood pressure, fibrinogen, triglycerides and in women also for menopausal status. Race and sex-specific correlations with serum total sialic acid for selected cardiovascular risk markers. RESULTS: The entire sialic acid distributions were shifted to the right in Caucasian men and women compared to Japanese men and women. The mean +/- standard deviation concentrations of S-TSA were 54.1 +/- 5.3 mg/dl in Japanese men and 58.7 +/- 5.6 mg/dl in Caucasian men (P < 0.001). In women, the concentrations were 54.8 +/- 5.1 and 63.1 +/- 6.0 mg/dl respectively (P < 0.001). S-TSA level correlated significantly and positively with fibrinogen levels in Caucasian and Japanese men and women and with triglyceride levels in Caucasian and Japanese men and in Caucasian women but not in Japanese women. After adjustment for age, systolic blood pressure, fibrinogen, triglycerides and menopausal status, the sialic acid levels were 2.2 (P = 0.009) and 6.2 (P < 0.001) mg/dl higher in Caucasian compared to Japanese men and women respectively. CONCLUSIONS: Higher S-TSA levels in Caucasians living in Minneapolis compared to Japanese living in Akita, Japan is in concordance with the higher cardiovascular mortality in the US. Differences in S-TSA levels may reflect international differences in the prevalence of atherosclerosis.

Use of monoclonal antibodies for weak affinity chromatography.

Weak monoclonal antibodies were used as ligands in a high-performance liquid affinity chromatography system. Isocratic weak affinity chromatography was achieved when similar carbohydrate antigens were separated according to their weak binding to the immobilized monoclonal antibody. These chromatographic systems were studied in detail in terms of affinity, specificity and efficiency. The influence of the physico-chemical factors of temperature, pH, ionic strength and organic solvents was also evaluated. The issue of specificity was specially considered, as non-specific interactions are prevalent and usually of a weak affinity nature. This study clearly demonstrates the potential to use weak affinity biological interactions as the basis of chromatographic analysis and separation.

The association between serum sialic acid and asymptomatic carotid atherosclerosis is not related to antibodies to herpes type viruses or Chlamydia pneumoniae. The Atherosclerosis Risk in Communities (ARIC) Study Investigators.

BACKGROUND: Total serum sialic acid is a recently investigated marker for cardiovascular mortality and carotid atherosclerosis. This study tested the hypothesis that past infection by Herpes simplex type 1 or type 2 viruses or Cytomegalovirus or Chlamydia pneumoniae accounts for the association between serum total sialic acid and atherosclerosis. METHODS: Population-based samples of men and women living in four US communities were used in a cross-sectional study. Cases and matched controls were defined by B-mode ultrasound measurements of carotid and popliteal arterial wall thickness. In all, there were 267 case control pairs with information about antibody titres to viruses and 256 pairs with information about antibody titres to Chlamydia pneumoniae. RESULTS: Serum total sialic acid (S-TSA) level was significantly higher in cases with carotid atherosclerosis compared to their controls. The odds ratio for carotid atherosclerosis associated with sialic acid level above 75th percentile was 1.73 (95% confidence interval [CI]: 1.02-2.95) in the sample with information about antibodies to viruses and 1.70 (95% CI: 1.00-2.93) in the sample with information about antibodies to C. pneumoniae. Adjustment for titres of antibodies to viruses and C. pneumoniae had no impact on the relation between sialic acid and carotid atherosclerosis. CONCLUSIONS: From these results, it seems unlikely that previous infection by any of these micro-organisms accounts for the relation between S-TSA level and carotid atherosclerosis.

Association between serum sialic acid concentration and carotid atherosclerosis measured by B-mode ultrasound. The ARIC Investigators. Atherosclerosis Risk in Communities Study.

BACKGROUND: Previous studies have shown that the serum level of sialic acid is associated positively with mortality from coronary disease and stroke. In this study its relation with carotid atherosclerosis was evaluated. METHODS: From the Atherosclerosis Risk in Communities (ARIC) Study, 323 cases with carotid intima-media wall thickness above the 90th percentile (measured with B-mode ultrasound) were matched 1:1 with controls without atherosclerosis. Serum sialic acid, plasma LDL and HDL cholesterol, serum insulin concentrations, blood pressure, antihypertensive medication use, and smoking status were used to assess the independent contribution of the sialic acid level to carotid atherosclerosis. RESULTS: The mean (SD) serum sialic acid concentration was 75.0 (9.7) mg/dl in cases and 70.7 (8.9) mg/dl in controls (P = 0.0001). In a conditional logistic model with adjustment for age, LDL-cholesterol, HDL-cholesterol, serum insulin, smoking and hypertension, the odds ratio associated with sialic acid above the 75th percentile (> or = 78.3 mg/dl) versus below was 1.65 with a 95% confidence interval of 1.01-2.70. CONCLUSION: The sialic acid level is correlated with the presence of carotid atherosclerosis, independently of major cardiovascular disease risk factors. The biological mechanism behind this association is not resolved.

E-selectin: sialyl Lewis, a dependent adhesion of colon cancer cells, is inhibited differently by antibodies against E-selectin ligands.

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe(x))/ Sialyl Lewis a (SLe(a)) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe(x) and antibodies directed against related Lewis epitopes, Le(x) and Le(a), had no significant effect on adhesion. Three antibodies directed against SLe(a) differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe(a) structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe(a) present on individual proteins, suggesting that presence and right presentation of SLe(a) epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe(a) interaction. SLe(x) and related epitopes, Le(x) and Le(a), seem to have limited relevance for colon cancer cell recognition of E-selectin.