The role of Concanavalin A-binding fraction in cholesterol crystallization in native human bile

Background/Aims: Many Concanavalin A-binding glycoproteins have been proposed to influence cholesterol crystallization in human bile. This has been studied mainly by addition of the Concanavalin A-binding fraction to model bile. The physiological relevance of the proteins in native bile is not yet known. The aim of this study was to establish the role of the Concanavalin A-binding fraction in cholesterol crystallization in native human gallbladder bile.Methods: To determine the effects of the removal of Concanavalin A-binding fraction, fresh human gallbladder bile was incubated with either Concanavalin A-Sepharose or Sepharose alone. Beads were sedimented and crystallization was studied in the supernatant.Results: Extraction of Concanavalin A-binding fraction decreased crystallization in fast-nucleating biles (Crystal Detection Time <-4 days). Slow-nucleating biles were not affected. The effect could not be related to the content of known pronucleating proteins (IgA, IgM, haptoglobin, aminopeptidase N and α1-acid-glycoprotein), since the slow-nucleating biles contained similar amounts of these proteins.Conclusions: Although Concanavalin A-binding fraction always accelerated crystallization when added to model bile, removal of the same fraction from native bile often had no effect. We conclude that slow-nucleating biles in particular contain undertermined factors which regulate the activity of pronucleators.     

Characterization of the cholesterol crystallization-promoting low- density particle isolated from human bile

BACKGROUND & AIMS: Biliary concanavalin A-binding glycoprotein (CABG) contains cholesterol crystallization-promoting activity that is not accounted for by the pronucleators that have been characterized in this fraction. The aim of this study was to isolate and characterize the missing activity. METHODS: Biliary glycoprotein was isolated using concanavalin A-Sepharose. Promoting activity in CABG was purified using density gradient ultracentrifugation. RESULTS: Activity in CABG separated into two fractions at low (1.08) and high (1.29) density, which showed different crystallization kinetics in a model bile. The high-density fraction had a late onset time (49.2 +/- 17.8 hours) but a high crystal growth rate (13.4 +/- 5.2 micrograms. mL-1.h-1). The low- density fraction had a rapid onset time (33.9 +/- 20.9 hours) but a slower growth rate (6.5 +/- 3.8 micrograms.mL-1 .h-1). The high-density fraction was not further characterized in this study. The low-density fraction contained solid particles consisting of lipid and very little protein, and the activity was fully pronase resistant. Delipidation of the low-density fraction removed all activity. CONCLUSIONS: A potent pronase-resistant nucleation-promoting activity was activated from human bile and characterized. The low-density fraction may be responsible for the rapid nucleation in bile from typical patients with fast-nucleating gallstones. (Gastroenterology 1996 Jun;110(6):1936-44)     

Immunoassay of acute phase reactants and Latex-CRP as activity tests in chronic staphylococcal osteomyelitis

20 patients with chronic staphylococcal osteomyelitis were tested for 6 acute phase reactants: alpha 1-antitrypsin, orosomucoid, haptoglobin, ceruloplasmin, C-reactive protein (CRP) and antichymotrypsin during different phases of the disease. CRP was best correlated to clinical activity and in 3 cases CRP and ceruloplasmin increased a few weeks before a clinically apparent exacerbation of the osteomyelitis took place. A Latex-CRP slide method showed fairly good agreement with CRP assessed by immunoassay. Determination of CRP is a suitable test for following the activity of chronic osteomyelitis.     

Serum protease inhibitors in opisthorchiasis, hepatoma, cholangiocarcinoma, and other liver diseases

Serum alpha 1-antitrypsin, alpha 1-antichymotrypsin and alpha 2-macroglobulin increased significantly in patients suffering from liver diseases: hepatoma, amoebic liver abscess, hepatitis, hepatic cirrhosis, cholangiocarcinoma, carcinoma of the head of pancreas including liver fluke infection (opisthorchiasis). Marked increase of alpha 1-antitrypsin and alpha 1-antichymotrypsin were found in cholangiocarcinoma, carcinoma of the head of pancreas, amoebic liver abscess, hepatic cirrhosis and hepatoma. alpha 2-macroglobulin increased markedly in hepatic cirrhosis. The concentrations of protease inhibitors found in opisthorchiasis were only moderately elevated.     

Carcinoembryonic antigen-related cell adhesion molecule 1 is the 85-kilodalton pronase-resistant biliary glycoprotein in the cholesterol crystallization promoting low density protein-lipid complex.

A pronase resistant 85-kd glycoprotein in the Concanavalin A-binding fraction (CABF) of biliary glycoproteins has been reported to act as a promotor of cholesterol crystallization. De Bruijn et al. (Gastroenterology 1996;110:1936-1944) found this protein in a low-density protein-lipid complex (LDP) with potent cholesterol crystallization promoting activity. This study identifies and characterizes this protein. An LDP was prepared from CABF by discontinuous gradient ultracentrifugation. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), blotting and immunochemical staining with anti-carcinoembryonic antigen, CEA-related adhesion molecule 1 (CEACAM1) cross-reacting antibodies. Biliary concentrations of CEA cross-reacting proteins were determined in patients with and without gallstones. Two isoforms of CEACAM1 (85- and 115-kd bands), CEA and 2 CEA cross-reacting protein bands of 40 and 50 kd were found in human bile. All bands were also present in CABF, but only a subfraction of the 85-kd band found in the LDP was resistant to digestion with pronase. CEACAM1-85 exhibited potent cholesterol crystallization promoting activity in vitro and accounted for most of the activity in CABF. Total CEA cross-reacting protein concentrations were the same in gallbladder biles from patients with cholesterol and pigment gallstones but only half of those in biles from nongallstone subjects. In conclusion, we have identified the protein component of the cholesterol crystallization promoting LDP to be CEACAM1-85.     

alpha 1-antitrypsin TAQ I polymorphism and alpha 1-antichymotrypsin mutations in patients with obstructive pulmonary disease.

Obstructive pulmonary disease is a multifactorial condition deriving from the interaction of environmental and genetic factors. From biochemical knowledge of the basis of the disease, alpha 1-antitrypsin and alpha 1-antichymotrypsin are considered two likely candidate genes. We therefore designed an association study comprising 232 unrelated Italian individuals divided as follows: 89 individuals with obstructive lung disease (66 with COPD and 23 with disseminated bronchiectasis) and 143 controls (45 patients with non-obstructive lung disease and 98 healthy individuals). We screened for Taq I (G1237A) polymorphism of the alpha 1-antitrypsin gene as well as the rare variants Bonn-1 (Pro229Ala), Bochum-1 (Leu55Pro), Isehara-1 (Met389Val) and Isehara-2 (1258delAA), and the common signal peptide polymorphism Thr-15Ala of the alpha 1-antichymotrypsin gene. The frequencies of Taq I G1237A alleles were 11.7 and 10.8% in obstructed patients and controls, respectively (P = 0.43), while those of signal peptide Thr-15Ala alleles were 51.6 and 50.3% in obstructed patients and controls, respectively (P = 0.42). We conclude that alpha 1-antitrypsin Taq I polymorphism and alpha 1-antichymotrypsin Thr-15Ala mutation are not major genetic risk factors for the development of obstructive lung disease in Italian patients. The alpha 1-antichymotrypsin rare variants were not detected: our results do not exclude the possibility that other alpha 1-antichymotrypsin gene mutations might be present in Italian obstructed patients but, if so, these genetic defects must be rare.     

Protease inhibitors in rheumatoid synovial fluid: a quantitative analysis

The concentrations of the main endogenous inhibitors of granulocyte proteases (anti-leukoprotease, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and alpha 2-macroglobulin) were estimated in paired samples of synovial fluid and serum/plasma from seropositive rheumatoid arthritics and controls. Rheumatoid synovial fluid contained significantly higher levels of all inhibitors except antileukoprotease. The influence of the synovial membrane on these concentrations was taken into account by comparing the ratio between the observed concentration and that predicted from a certain regression curve fitted to a set of non-inhibitory reference proteins of extra-articular origin (orosomucoid, albumin, and ceruloplasmin). Divergences were interpreted as the net result of intra-articular production or consumption of the inhibitor in question. The results suggested a consumption of antileukoprotease and alpha 1-antitrypsin in the rheumatoid joint, while the increased levels of alpha 1-antichymotrypsin and alpha 2-macroglobulin probably reflected the altered trans-synovial membrane protein flux with some reservation for alpha 2-macroglobulin.     

Serum concentration of 19 serum proteins in Crohn's disease and ulcerative colitis

The serum concentration of 19 serum proteins was determined by electrophoresis in 42 patients with Crohn's disease and 36 patients with ulcerative colitis. The results were compared with 78 healthy persons as matched controls. Distinctive, but similar, changes were present in the two diseases. An increased serum concentration of orosomucoid, alpha(1)-antitrypsin, easily precipitable glycoprotein, alpha(1)-antichymotrypsin, haptoglobin, and haemopexin was present. The serum concentration was decreased for prealbumin, albumin, alpha(2)-HS glycoprotein, caeruloplasmin, alpha(2)-macro-globulin, and transferrin. No significant difference between the two diseases existed as far as the serum protein pattern was concerned.Certain proteins, ;the acute phase reactants' (orosomucoid, alpha(1)-antitrypsin, alpha(1)-antichymotrypsin, and haptoglobin) and the immunoglobulins were clinically useful, since their serum concentration reflected the grade of activity of the disease. A pronounced elevation of haptoglobin compared with that of the other ;acute phase reactants' was present in patients with Crohn's disease complicated by suppurative fistulas or abscesses. Patients with active Crohn's disease who responded favourably to medical treatment had significantly higher immunoglobulin levels than patients not responding. A similar observation, though not statistically significant, was made in patients with ulcerative colitis.     

Proteinase inhibitors in rheumatoid arthritis.

The concentrations of five normally occurring protease inhibitors in serum and synovial fluid were compared in patients with rheumatoid arthritis, osteoarthrosis, and normal controls. The patients with rheumatoid arthritis showed a significant rise in alpha1-antitrypsin, alpha1-antichymotrypsin, and inter-alpha-trypsin inhibitor (in decreasing order) in serum as well as in synovial fluid. In synovial fluid the inhibitors were present in their native form and bound to hyaluronate. A large molecular protein with immunological specificity of alpha1-antitrypsin, presumably a complex of alpha1-antitrypsin and a protease, could be shown in synovial fluid of all patients with classical and probable rheumatoid arthritis and not in that of the other subjects studied.23Author     

Protease inhibitors in patients with chronic obstructive pulmonary disease: the alpha-antitrypsin heterozygote controversy.

A group of 163 patients with chronic obstructive pulmonary disease, from the pulmonary service of a large urban hospital, were evaluated for their protease inhibitor (Pi) type by starch gel and crossed immunoelectrophoresis, for serum concentrations of alpha1-antitrypsin and alpha1-antichymotrypsin, and for pulmonary function. Of the patients with emphysema, 17.8% were of Pi type Z; 50% of these were less than 45 years of age, compared to 13% of those of Pi type M. Of all patients with chronic obstructive pulmonary disease, 4.9% were of Pi type Z; 4.9% of patients were of Pi type MZ (heterozygotes) compared with 1.9% of the control population. There was an increased incidence of chronic obstructive pulmonary disease in persons of Pi type MZ, but no increase in persons of Pi type MS. Concentrations of both alpha1-antitrypsin and alpha1-antichymotrypsin were increased and were correlated. No patient had a deficiency of alpha1-antichymotrypsin.     

Human aortic intima protein composition during initial stages of atherogenesis

Protein extracted from 24 human aortic intimas (6-33 years old) with 9 M urea mixture, were studied after separation by two-dimensional gel electrophoresis (2-DE) and silver staining. The protein composition of normal intima in 4 cases, each without any gross changes in the thoracic aorta, displayed similarity. In each 2-DE protein pattern of these intimas about 150 polypeptide spots were detectable/mg of wet tissue. Major and medium polypeptides were described by relative molecular weight Mr in kilodaltons (kDa) and relative charge Cr. Major proteins found were actin (P44-18; Mr = 44 kDa; Cr = -18), tropomyosin-like proteins (P34-29, P35-28.5, P36-31) and two glycoproteins (G35-21, G35-23.5). Several new major and medium extracellular proteins were demonstrated in fibro-fatty lesions as well as in the lesion-free intimas adjacent to lesion in 3 cases. Many of these proteins appeared to originate from plasma: albumin, IgG, alpha 1-antitrypsin, transferrin, haptoglobin beta-chain, apo A-I, apo A-II, fibrinogen beta-chain, alpha 2-HS glycoprotein and alpha 1-antichymotrypsin. Visual comparison of intimal protein patterns from 17 different cases with varying degree of fatty streaks in the thoracic aorta, showed variability in 2 polypeptides P32-17.8 and P32-19.8 as well as 4 plasma proteins albumin, alpha 1-antitrypsin, transferrin and apo A-I. This study suggests that changes in protein composition may occur in the human aortic intima during the initial histological stages of atherogenesis providing potentially useful markers for their identification and pathophysiological evaluation.     

Human inhibitor of the first component of complement, C1: characterization of cDNA clones and localization of the gene to chromosome 11.

C1 inhibitor is a heavily glycosylated plasma protein that regulates the activity of the first component of complement (C1) by inactivation of the serine protease subcomponents, C1r and C1s. C1 inhibitor cDNA clones have been isolated, and one of these (pC1INH1, 950 base pairs) has been partially sequenced. Sequence analysis demonstrates that the C1 inhibitor is a member of the serpin "superfamily" of protease inhibitors. In the region sequenced, C1 inhibitor has 22% identity with antithrombin III, 26% with alpha 1-antitrypsin and alpha 1-antichymotrypsin, and 18% with human angiotensinogen. C1 inhibitor has a larger amino-terminal extension than do the other plasma protease inhibitors. In addition, inspection of residues that are invariant among the other protease inhibitors shows that C1 inhibitor differs at 14 of 41 of these positions. Thus, it appears that C1 inhibitor diverged from the group relatively early in evolution, although probably after the divergence of angiotensinogen. Southern blot analysis of BamHI-digested DNA from normal individuals and from rodent-human somatic cell hybrid cell lines (that contain a limited but varied human chromosome complement) was used to localize the human C1 inhibitor gene to chromosome 11.     

Acute phase proteins and liver allograft rejection

We studied the sequential changes in the positive acute phase reactants orosomucoid, alpha 1-antichymotrypsin, alpha 1-antitrypsin, haptoglobin and C-reactive protein, and in the negative acute phase reactant, prealbumin, as well as in alpha 2-macroglobulin and beta 2-microglobulin in serum following orthotopic liver transplantation in nine adult patients. Synchronous increases in all five positive acute phase reactants were observed in association with acute liver rejection episodes; the elevations were statistically significant for orosomucoid and alpha 1-antichymotrypsin. In four out of nine well-documented rejection episodes C-reactive protein did not increase at all. Marked variations occurred in post-transplant prealbumin and alpha 2-macroglobulin levels; both proteins tended to decrease 2-3 days after the rejection was diagnosed. The circulating levels of beta 2-microglobulin were raised during the post-transplant period and markedly increased in association with acute graft rejection episodes. The results demonstrate marked changes in plasma protein concentrations in association with acute liver allograft rejection episodes; the kinetics of serum acute phase reactants is very similar to that found in inflammation and tissue injury in general.     

Gallstone formation. Local factors

Bile supersaturation is necessary for cholesterol gallstones to form. Not all people with supersaturated bile form gallstones, however, and additional factors must be present. The role of pronucleating substances has been extensively studied. Of these, proteins, especially mucin, are best understood. Mucin is secreted by the gallbladder epithelium and may act as a nidus for crystal nucleation. Other proteins that may act as pronucleators include alpha 1-acid glycoprotein, alpha 1-antichymotrypsin, phospholipase C, and a small calcium binding protein. The role of antinucleating factors is less well understood. Certain drugs, including octreotide and ceftriaxone, may also predispose to stone formation. Another local factor is gallbladder stasis, a well-known risk factor for pigment stone formation. More recent research has focused on the role of bacterial infection, which has long been believed to be a factor in pigment gallstone formation. Newer data also support a role for infection in cholesterol gallstone pathogenesis. Additionally, genetic factors that may predispose a patient to cholesterol gallstones have been identified in mice and in humans.     

Cloning and sequence of cDNA coding for alpha 1-antitrypsin.

Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood. One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor. It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides. Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous. A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs. This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin. The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.     

Improvement of CDG diagnosis by combined examination of several glycoproteins

Congenital disorders of glycosylation (CDG) represent a family of genetic diseases with broad clinical presentation. Initial diagnosis is currently mainly based on the identification of hyposialylated serum transferrin (TF) by isoelectric focusing (IEF). To improve the diagnosis of known CDG types and to identify so far unknown CDG cases, additional glycoproteins, alpha1-antitrypsin titrypsin (alpha1-AT) and alpha1-antichymotrypsin (alpha1-ACT), were studied. According to the patterns of transferrin, enzyme assays and mutation analysis, 16 patients with various clinical symptoms suspicious for CDG were divided into three groups: group A (n = 6) with confirmed CDG; group B (n = 4) with clear abnormal TF-IEF patterns of unknown origin (all known CDG types were excluded) and group C (n = 6) with borderline TF-IEF patterns; 164 samples served as a control group. Automated IEF of TF, alpha1-AT and alpha1-ACT was carried out using a PhastSystem. CDG patients with glycosylation defects of known origin (group A) and patients with abnormal TF-IEF patterns due to glycosylation defects of as yet unknown origin (group B) showed abnormal IEF patterns of all three glycoproteins. These results confirmed generalized defects of glycosylation. Furthermore, the IEF pattern of alpha1-ACT seems to allow a differentiation between CDG Ia and CDG Ic. However, patients with borderline TF-IEF pattern (group C) showed a normal alpha1-AT-IEF pattern. Four of these six patients also showed a normal alpha1-ACT-IEF pattern; this constellation suggests that CDG can most likely be excluded. In the two remaining patients of group C with a borderline TF-IEF pattern an abnormal pattern of alpha1-ACT-IEF was obtained which needs further investigations. We conclude that the combined investigation of three glycoproteins provides additional information in the diagnostic work-up of patients with possible CDG. The suspicion of CDG in patients with apparent glycosylation defects of unknown origin or borderline TF-IEF pattern can be either substantiated or weakened.     

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.     

Correlations between clinical activity, endoscopic severity, and biological parameters in colonic or ileocolonic Crohn's disease. A prospective multicentre study of 121 cases. The Groupe d'Etudes Thérapeutiques des Affections Inflammatoires Digestives.

The relationships between clinical activity, endoscopic severity, and biological parameters in Crohn's disease have not been thoroughly investigated and a link was therefore sought between these three elements. The following parameters were determined simultaneously in 121 consecutive patients with colonic or ileocolonic Crohn's disease: Crohn's disease activity index, Crohn's disease endoscopic index of severity, and serum albumin, alpha 2-globulin, alpha 1-antitrypsin, orosomucoid, C reactive protein, erythrocyte sedimentation rate, platelets, lymphocyte and polymorphonuclear cell counts, haematocrit, and faecal alpha 1-antitrypsin concentration. The distribution of these parameters was studied and transformation was used so that data matched the normal distribution closely. A weak but significant correlation (r = 0.32; p < 0.001) was found between clinical and endoscopic indices in the whole group of patients and this correlation seemed to be homogenous in various patient subgroups (clinically quiescent or active disease, pure colonic disease, untreated patients). Endoscopic or clinical indices were also found to be weakly linked with biological parameters (r < 0.50). Stepwise linear regression identified C reactive protein as predictive of the clinical index, and, successively, alpha 2-globulin, erythrocyte sedimentation rate, faecal alpha 1-antitrypsin, serum orosomucoid, and alpha 1-antitrypsin as predictive of the endoscopic index. Both predictions were poor--the biological variables accounting for only 22 and 44% respectively of the clinical and endoscopic index variations. In conclusion, Crohn's disease clinical activity seems to be virtually independent of the severity of the mucosal lesions and biological activity.     

Faecal clearance of α1-antitrypsin reflects disease activity and correlates with rapid turnover proteins in chronic inflammatory bowel disease

Faecal clearance of alpha 1-antitrypsin was measured in patients with ulcerative colitis and Crohn's disease and compared with disease activity and markers of protein-calorie malnutrition. Patients with active ulcerative colitis and Crohn's disease showed elevated clearance of alpha 1-antitrypsin and clearance declined in most patients with induction of remission. However, even with inactive disease, elevated protein loss persisted in some patients, presumably reflecting residual inflammation in the intestinal mucosa. There was a significant correlation between clearance of alpha 1-antitrypsin and serum levels of retinol-binding protein and transferrin in patients with ulcerative colitis and with retinol-binding protein in patients with Crohn's disease. Clearance of alpha 1-antitrypsin reflects disease activity in inflammatory bowel disease and correlates with serum levels of rapid-turnover proteins such as retinol-binding protein and transferrin, which are markers for the presence of protein-calorie malnutrition.     

The elimination of aged glycoproteins by the isolated perfused rat liver

The isolated livers of Sprague-Dawley rats aged ten months were perfused with nine different glycoproteins (coeruloplasmine, haptoglobin, transferrin, alpha2-mocroglobulin, apolipoprotein A, plasminogen, acid alpha1-glycoprotein, alpha1-antitrypsin, beta2-glycoprotein), which were suspended in a basic medium free of hemoglobin and other proteins. Besides a control group two other groups were formed to give a model for aging: one group of glycoproteins was desialysed, another was desilysed and incubated with immunoglobulins before the perfusion. In spite of the application of two procedures which are supposed to produce a model of old glycoproteins, neither treatment resulted in the recognition, binding and elimination of the glycoproteins from the perfusion medium.