Characterization and evaluation of immunochemical methods for the measurement of fecal alpha 1-antitrypsin

Measurement of alpha 1-antitrypsin in feces has been proposed as a method of diagnosing a protein-losing enteropathy. This approach makes use of an endogenous marker rather than radioisotopically labeled materials such as 51CrCl3 or 131albumin to measure protein clearance. The validity of using fecal alpha 1-antitrypsin measurement as a reflection of protein loss through the gastrointestinal tract has been demonstrated by several investigators. The authors report here the characterization of excreted alpha 1-antitrypsin and an evaluation of the immunochemical methods used to measure this protein. They find alpha 1-antitrypsin to be excreted both as a protease-antiprotease complex and in a form that is relatively unaltered compared with serum alpha 1-antitrypsin. The proportion of alpha 1-antitrypsin excreted as a complex was found to vary from patient to patient. Formation of the protease-antiprotease complex was found to decrease the apparent alpha 1-antitrypsin concentration when radial immunodiffusion or immunonephelometry were used. The observed bias was greater for radial immunodiffusion. When these methods were applied to a newborn population at risk for necrotizing enterocolitis, radial immunodiffusion was found to have better sensitivity and a higher predictive value for a positive result than the nephelometric method. The use of fecal alpha 1-antitrypsin for diagnosis of protein-losing enteropathy appears to be best accomplished by radial immunodiffusion.     

Mutation in the 3'' region of the alpha-1-antitrypsin gene and chronic obstructive pulmonary disease.

A mutation in the 3' flanking region of the alpha-1-antitrypsin gene has been reported to be associated with chronic obstructive pulmonary disease (COPD). We have investigated the prevalence of this mutation in a group of 185 patients with airway obstruction and in 69 non-obstructed controls. The subjects were selected on the basis of their development of lung cancer and therefore had similar exposure to cigarette smoke, the major risk factor for COPD. In the majority of subjects, the level of airway inflammation and loss of elastic recoil was known, and therefore we were able to test whether the mutation was associated with one of these pathological mechanisms. The prevalence of heterozygotes for the mutation was 10% in both the obstructed group and controls. The mutation was not associated with increased airway inflammation or loss of elastic recoil. This result indicates that the 3' mutation is not a significant risk factor for COPD in this population, and suggests heterogeneity in the pathogenesis of the disease.     

Hepatocytic PAS-positive diastase-resistance inclusions in the absence of alpha-1-antitrypsin deficiency--high prevalence in alcoholic cirrhosis

The presence of PAS-positive, diastase-resistant inclusions in the cytoplasm of the hepatocytes is characteristic of alpha-1-antitrypsin deficiency. The purpose of this investigation was to determine whether the presence of these inclusions is a specific feature, permitting the recognition of alpha-1-antitrypsin deficiency in patients with liver disease. We examined the liver specimens from 20 patients suffering from alcoholic cirrhosis with the Pi M phenotype, i.e., in whom alpha-1-antitrypsin deficiency was excluded. In seven of these patients, PAS-positive, diastase-resistant inclusions were seen in the hepatocytes; in two patients, these inclusions contained a material antigenically similar to alpha-1-antitrypsin. These inclusions might represent deposits of glycoproteins poorly excreted by the diseased hepatocytes. It is concluded that, in patients with liver disease, the presence of PAS-positive, diastase-resistant inclusions--even containing alpha-1-antitrypsin--in the cytoplasm of the hepatocytes does not permit the hepatic lesions to be ascribed to alpha-1-antitrypsin deficiency.     

Calcium metabolism evaluated by 47calcium-kinetics: a physiological model with correction for faecal lag time and estimation of dermal calcium loss

Sixty-two calcium balance and 47Ca-turnover studies were performed in 51 individuals to evaluate the accuracy and the sensitivity of the methods. The data were analysed according to a modification of the expanding calcium pool model using an improved Bauer-Carlsson-Lindquist (BCL) formulation and an iterative computer procedure. A 7-day whole body retention curve (R1) combined with a retention curve constructed from excretion data alone (R2) was used to estimate dermal calcium loss (d) and to demonstrate the significance of individual corrections for delay in faecal excretion (faecal lag time = delta t). The mean d was 1.58 mmol Ca/day. The introduction of delta t improved the goodness of fit of the data to the model. delta t based on 47Ca-kinetics was superior to a fixed delta t of zero (P less than 0.01) or 2 days (P less than 0.05). The model derived renal calcium excretion rate was highly correlated (r = 0.98, P less than 0.001) to the chemical measured excretion rate. A similar highly significant correlation [RS = 0.78 (Spearman), P less than 0.001] was found between the model derived delta t and the carmine red delta t. These results indicate a high accuracy of the model. The directly measured parameters showed an excellent reproducibility with a coefficient of variation (CV) less than 4%. The reproducibility of the derived parameters was acceptable (CV = 10-20%) except for the balance (CV = 72%).     

Acute phase proteins in monoclonal gammapathies]

IL-6 is now recognized as a growth factor for plasma cells as well as a C Reactive Proteine inducer. This prompted a reappraisal of acute phase reactants in monoclonal gammapathies. Eight acute phase proteins were assayed in patients with multiple myeloma (n = 51), MGUS (n = 17) and Waldenstr?m's macroglobulinemia (n = 5). The CRP level was above 10 mg/l in 27% of all myeloma patients, in 39% of patients with active myeloma, in 4 of 5 patients with Waldenstr?m's macroglobulinemia and in none of the MGUS patients. Fibrinogen, alpha-1-antitrypsin and orosomucoid levels were significantly higher in the myeloma group than in the MGUS group. Differences were not significant for haptoglobin, ceruleoplasmin, transferrin, and alpha-2-macroglobulin. Serial assays in 22 myeloma patients showed that CRP levels were correlated with disease activity. A biologic inflammatory syndrome, defined as a significant variation in two or more acute phase reactants, was demonstrated in 41% of myeloma patients, 18% of MGUS patients, and 60% of Waldenstr?m's macroglobulinemia patients. Active disease was significantly more common among myeloma patients with biologic evidence of inflammation, as compared with myeloma patients without biologic inflammation. These data suggest that similarly to IL-6 acute phase reactants are markers for disease activity in multiple myeloma.     

Immunohistochemistry of primary gastrointestinal lymphomas: a study of 76 cases

A retrospective study of 76 primary gastrointestinal lymphomas utilizing an avidin: biotinylated horseradish peroxidase complex (ABC) technique demonstrated 22 B-cell lymphomas, including two associated with alpha-heavy chain disease. Seven cases were classified as true histiocytic lymphomas based on a positive reaction for one or more of three histiocytic enzyme markers utilized, predominantly alpha-1-antitrypsin and alpha-1-antichymotrypsin. However, in 20 cases, an intense admixture of reactive histiocytes was noted and these cells stained preferentially for the enzyme, lysozyme. Twenty cases, which stained for both kappa and lambda light chains and positively or negatively for albumin, could not be classified and 27 cases failed to stain with any of the antisera utilized.     

MVarallo: a new M(Like) alpha 1-antitrypsin-deficient allele.

A 73-year-old never-smoker woman with chronic bronchitis, increasing dyspnoea, and airflow limitation with a FEV1 of 49% of predicted value had low serum level of alpha-1-antitrypsin (69 mg/dL, normal range 150-350). Isoelectric focusing showed an Mlike pattern. Direct sequencing showed, in the second exon, a particular DNA alteration localized between codon 41 and codon 51: a region of 30 base pairs (bp) was completely deleted and substituted by a 22-bp sequence. The resulting loss of 8 bp yields, in the second exon, a 70-71 stop codon. This new Mlike variant was denominated MVarallo from the site where it was discovered.     

Study of various serum proteins in lung cancer. Immunoglobulins A, G, M, haptoglobin, alpha-1-antitrypsin, alpha-2-macroglobulin]

A statistical study of the plasma concentration of immunoglobulin, alpha-1-antitrypsin, haptoglobin, and alpha-2-macroglobulin in 153 patients with primary carcinoma of the lung showed a strong increase in alpha-1-antitrypsin, haptoglobin and A and C immunoglobulins, whilst alpha-2-macroglobulin increases very moderately, and IgM does not vary. The relationships between variables are also modified, thus there appears a correlation between IgA and IgG to the detriment of the IgG/IgM relationship. Furthermore the coefficient of the correlation IgG/alpha-2-macroblobulin is lower in cancer patients.     

Ceruloplasmin can substitute for rabbit serum in stimulating the growth of Treponema denticola.

The growth of Treponema denticola in a complex medium was stimulated by human ceruloplasmin and, to a lesser extent, albumin, alpha-1-antitrypsin, alpha-1-acid glycoprotein, and alpha-2-macroglobulin. Human ceruloplasmin could stimulate growth of T. denticola as well as or better than 5% rabbit serum, with a dose-response relationship of between 1 and 5 mg of ceruloplasmin per ml of culture medium. These results suggest that ceruloplasmin could substitute for rabbit serum in stimulating the growth of T. denticola.     

Human atrial natriuretic peptide in patients with type 1 diabetes mellitus: is it related to the development of diabetic nephropathy?

Summary There is controversy as to whether increased plasma levels of human atrial natriuretic peptide (hANP) in patients with type 1 diabetes mellitus may contribute to the development of diabetic nephropathy. Therefore, we decided to conduct two studies to examine the relationship of hANP levels to urinary albumin excretion and blood pressure. In a cross-sectional study, 83 randomly selected type 1 diabetic patients were investigated. 19 of the patients had increased urinary albumin excretion. 45 healthy volunteers served as controls. In a longitudinal study, 19 type 1 diabetic patients were examined for one year at monthly intervals. An increased risk of eventually developing diabetic nephropathy was identified in 7 out of these patients by repeatedly revealing increased urinary albumin excretion. On the average, hANP levels were increased in type 1 diabetic patients in comparison to controls (P < 0.001). In both studies, hANP levels were positively related (P < 0.05) to mean arterial blood pressure. There was no correlation between hANP levels and metabolic control. hANP levels lay within normal range irrespective of normal or elevated urinary albumin excretion provided that mean arterial blood pressure was normal. In the longitudinal study, increased urinary albumin and alpha-1-microglobulin excretion preceded the increase in both hANP levels and mean arterial blood pressure. Although hANP levels were evidently not related to the disease mechanisms of early diabetic nephropathy, it is tempting to speculate that hANP may contribute to the vicious circle connecting diabetic kidney disease to hypertension once that its levels are increased by elevated blood pressure.Abbreviation hANP human atrial natriuretic peptide     

Quantitative study of alpha 1 antitrypsin and alpha 2 macroglobulin in the sputum]

A quantitative study of alpha-1-antirypsin and alpha-2-macroglobulin in the sputum showed an increase in both during attacks of asthma and in patients with chronic obstructive bronchitis. The levels were much lower in asthma patients during remissions, in emphysema and in patients with chronic simple bronchitis, without associated bronchospasm. The parallel variations in levels of alpha-1-antitrypsin, albumin and transferrin in the sputum, contrasts with the course of secretory IgA and are in favour of a transudation process of alpha-1-antitrypsin across the respiratory mucosa.     

Alpha-1-antitrypsin phenotypes in malignant lymphoma.

The alpha-1-antitrypsin (alpha 1 AT) Pi phenotypes have been determined by isoelectric focusing in a series of 228 patients having a histologically diagnosed malignant lymphoma and in 250 healthy controls. The Pi MZ phenotype occurred in 13 patients with lymphoma (5 . 8%) and in five of 250 healthy individuals (2%). Furthermore, one patient with a Pi SS and three patients with an abnormal unknown phenotype, migrating slower than Z, were found in the lymphoma group. No prevalence for a special lymphoma type was observed among the abnormal Pi phenotype patients. The increased incidence of abnormal Pi phenotypes in malignant lymphoma's support the hypothesis of the possible role of alpha 1 AT in development of immunopathological disorders.     

Alpha-1-antitrypsin (Pi) types in Down''s syndrome

Alpha-1-antitrypsin (Pi) phenotypes have been determined in 40 patients suffering from Down's syndrome. Thirty-six of the patients were found to have a normal M phenotype, whereas two deficient phenotypes of the MS variety were observed. In addition, two M variants were noted. The significance of an M variant phenotype in some patients with Down's syndrome is not completely understood and is currently under investigation. Since the majority of the patients had a normal alpha-1-antitrypsin phenotype, the results of this study indicate that a deficiency in alpha-1-antitrypsin plays no role in the respiratory fragility of individuals with Down's syndrome.     

Differences between sheep excreting sodium predominantly in their urine or in their faeces: the effect of changes in sodium intake.

Sheep receiving a total of about 31 mmol day-1 (0.5 mmol kg-1) of sodium were classified according to the predominant route of sodium excretion; urinary (U) or faecal (F). U sheep had a greater water turnover than F sheep; their intake was 41% higher and they produced 133% more urine but there was little difference in faecal water loss. Most faecal sodium was readily exchangeable with water in both groups. When sodium intake was reduced by 80% (to 6 mmol day-1; 0.1 mmol kg-1), the reduction in total sodium excretion was equally effective in F sheep and U sheep after 48 h and after 2 weeks the overall losses of sodium were smaller in F sheep. On sodium intakes close to requirement (0.1 mmol kg-1 or less) the majority of the sheep excreted most of their sodium in faeces and did so on intakes up to 0.5 mmol kg-1 day-1. Excess dietary sodium is mainly excreted renally. When sodium intake is increased abruptly (by 20 mmol day-1, 0.3 mmol kg-1), total sodium excretion only increases gradually but after about 3 days it 'overshoots' as in humans.     

Localization of CEA,HCG, Lysozyme,alpha-1-antitrypsin,and alpha-1-antichymotrypsin in gastric cancer and prognosis

Summary Carcinoembryonic antigen (CEA),-human chorionic gonadotropin (HCG), alpha-1-antichymotrypsin (ACT), alpha-1-antitrypsin (AAT) and Lysozyme (LYS) were traced by immunoperoxidase staining in gastric carcinomas. The immunohistological results were evaluated in relation to histological types (WHO and Laurén), stage of disease, grade and survival time. CEA was demonstrated in 96% of the tumours, HCG in 34%, ACT in 78%, AAT in 42%, and LYS in 71%. Comparing the staining patterns of the antigens and the intensity of staining some differences were notable. Except for signet-ring cell carcinomas, all of which were intensively positive, CEA expression decreased significantly with loss of differentiation. This observation was not seen with the other marker substances. None of the tested markers was characteristic for one particular histological type, nor could they be correlated with the tumour stage or grade. The marker positivity of CEA, ACT and LYS was not related to survival time. For HCG only, a correlation between tissue expression and a restricted survival time was established. Patients with AAT positive carcinomas had a significantly better survival probability.     

Periodic acid Schiff-positive non-glycogenic globules in hepatocytes. Differential diagnostic aspects in screening for alpha-1-antitrypsin globules in an autopsy material.

Thirty-eight subjects with diastase-resistant PAS-positive cytoplasmic globules in hepatocytes were found among 238 autopsies. In 15 of the 38 subjects the globules were antigenically alpha-1-antitrypsin, in 23 subjects they were not. The latter globules were found in centrilobular regions, the alpha-1-antitrypsin globules mainly in periportal regions. The non-alpha-1-antitrypsin globules showed less differences in size (6-10 mu) and a smaller number per hepatocyte (1-7) than the alpha-1-antitrypsin globules (1-40 mu and 1-30 per cell). The non-alpha-1-antitrypsin globules were only demonstrated in livers with centrilobular sinusoidal dilatation having, in all cases but one, also centrilobular, confluent necrosis. This type of globules can be assumed to be of differential diagnostic importance mainly in an autopsy material. The nature of these globules is discussed.     

Urinary zinc excretion and acute phase response in cancer patients

We investigated urinary zinc and serum levels of C-reactive protein, alpha-1 acid glycoprotein, haptoglobin, transferrin and prealbumin in 55 patients with solid tumors and 20 controls. Urinary zinc, serum C-reactive protein, alpha-1 acid glycoprotein and haptoglobin were significantly higher, and serum prealbumin was significantly lower in cancer patients. A significant positive correlation between urinary zinc and C-reactive protein, alpha-1 acid glycoprotein and haptoglobin, as well as a negative correlation with transferrin and prealbumin were observed. Hyperzincuria in cancer patients appears to be linked to the acute phase response. Our data provide further evidence implicating systemic inflammatory response in increased urinary zinc excretion. Correspondence to: B. Melichar     

Alagille syndrome associated with a paracentric inversion 20p12.2p13 disrupting the JAG1 gene

Mutations in the human gene Jagged1 (JAG1) localized in 20p12 have been recently identified as causal for the anomalies found in patients with Alagille syndrome (AGS). This gene encodes a ligand for the Notch1 transmembrane receptor, which plays a key role in cell-to-cell signaling during differentiation and is conserved from C. elegans to human. We report a paracentric inversion (PAI) of chromosome 20p12.2p13 in an individual with AGS who also had alpha-1-antitrypsin deficiency. To our knowledge, this is the first published case of PAI involving the short arm of chromosome 20. Using FISH, fiberFISH, and molecular studies with a approximately 40 kb cosmid clone encompassing the entire 36 kb JAG1 gene, we demonstrate that the gene was disrupted by the inversion breakpoint between exons 5 and 6. An unusual association between two most common causes of chronic liver disease in childhood, AGS and alpha-1-antitrypsin deficiency, as well as their influence on the proband's abnormal phenotype are discussed.     

Alpha-1-antitrypsin hepatic cell globules in autotransplanted liver of rats.

The autotransplantation of liver into the subcutaneous tissues in rats and hamsters resulted in the development of periodic acid Schiffs staining positive granules, which were resistant to diastase digestion. These granules were confirmed as alpha-1-antitrypsin by immunohistochemical staining. The appearance of the alpha-1-antitrypsin containing hepatic granules is comparable to those seen in the liver of humans with genetically determined alpha-1-antitrypsin deficiency.     

Macrophages and mast cells in chronic cholecystitis and "normal" gall bladders

An attempt was made to quantify mast cells using toluidine blue and macrophages, with alpha-1-antitrypsin as a marker, from adjacent sections in the mucosa of two groups of gall bladders showing either minimal inflammatory change or established chronic cholecystitis. The results were expressed as cells/mm2 of mucosa. Alpha-1-antitrypsin showed both macrophages and mast cells, and therefore an estimate of macrophage numbers was obtained by subtraction. Mast cells comprised more than 60% of the alpha-1-antitrypsin positive cells. There were significantly (p greater than 0.001) more mast cells and macrophages in minimal inflammatory gall bladder mucosa than in established chronic cholecystitis.