Serum AAT Research Articles

Genotyping of α-antitrypsin in ten Croatian families

Objectives: The aims of the study were to determine α-antitrypsin (AAT) genotype by a simple DNA-based method and to investigate the association of AAT genotype and serum AAT concentration in a group of ten families. Methods and results: AAT genotype was determined by PCR-RFLP and serum concentration by radial immunodiffusion in samples from each member of ten families (mother, father, and child/children). In the group of parents, five normal genotypes, Pi MM, with a normal serum AAT concentration, and fifteen Pi MZ genotypes, four of them with slightly decreased (43%–66% of the mean) AAT concentration were detected. In the group of children, particular genotypes followed the mode of inheritance. There were eight Pi MZ, three of them with slightly decreased (52%–60% of the mean) AAT concentration, and five Pi ZZ genotypes with considerably decreased (24%–45% of the mean) AAT concentration. Conclusions: PCR-RFLP is the method of choice for AAT genotyping. AAT concentration is not a reliable biochemical marker of AAT deficiency. Determination of AAT genotype in family studies allows the risk of deficient allele inheritance to be followed up and assessed. Early diagnosis of a deficient AAT genotype contributes to the success of currently widely available AAT replacement therapy.

Hydrophobic interaction chromatography of human serum α 1-antitrypsin and α 1-acid glycoprotein

The relative hydrophobicity of human serum α 1-antitrypsin (AAT) and α 1-acid glycoprotein (AGP) in comparison to various reference proteins was determined by hydrophobic interaction chromatography (HIC). Apolar character of glycoproteins was generated using three different cosmotropic salts, ammonium sulfate, sodium sulfate, sodium citrate and isocratic, or reversed linear gradient elution techniques. Human serum AAT and AGP showed different apolar properties on Fractogel EMD phenyl and propyl columns modulated either by the type and concentration of cosmotropic salts, or by the pH of the mobile phase. According to its higher carbohydrate content AGP proved to be more polar than AAT. Human serum AAT and AGP were pre-separated by Cibacron Blue F3G-A dye ligand affinity chromatography and based on their different hydrophobicity were fractionated and purified by HIC.

Evidence for excessive bronchial inflammation during an acute exacerbation of chronic obstructive pulmonary disease in patients with alpha(1)-antitrypsin deficiency (PiZ).

Patients with homozygous (PiZ) alpha(1)-antitrypsin (AAT) deficiency have not only low baseline serum AAT levels (approximately 10 to 15% normal) but also an attenuated acute phase response. They are susceptible to the development of premature emphysema but may also be particularly susceptible to lung damage during bacterial exacerbations when there will be a significant neutrophil influx. The purposes of the present study were to assess the inflammatory nature of acute bacterial exacerbations of chronic obstructive pulmonary disease (COPD) in subjects with AAT deficiency, to compare this with COPD patients without deficiency, and to monitor the inflammatory process and its resolution following appropriate antibacterial therapy. At the start of the exacerbation, patients with AAT deficiency had lower sputum AAT (p < 0.001) and secretory leukoprotease inhibitor (SLPI; p = 0.02) with higher elastase activity (p = 0.02) compared with COPD patients without deficiency. Both groups had a comparable acute phase response as assessed by C-reactive protein (CRP) but the AAT-deficient patients had a minimal rise in serum AAT (to < 6 microM). After treatment with antibiotics, in patients with AAT deficiency, there were significant changes in many sputum proteins including a rise in SLPI levels, and a reduction in myeloperoxidase (MPO) and elastase activity (p < 0. 005 for all measures); the sputum chemoattractants interleukin-8 (IL-8) and leukotriene B(4) (LTB(4)) fell (p < 0.01), and protein leak (sputum/serum albumin ratio) became lower (p < 0.01). The changes were rapid and within 3 d of the commencement of antibiotic therapy the biochemical markers had decreased significantly, but took a variable time thereafter to return to baseline values. In conclusion, patients with AAT deficiency had evidence of increased elastase activity at the start of the exacerbation when compared with nondeficient COPD patients which probably reflects a deficient antiproteinase screen (lower sputum AAT and SLPI). The increased bronchial inflammation at presentation resolved rapidly with 14 d of antibiotic therapy.

Multiplex PCR assay for the detection of genetic variants of alpha1-antitrypsin.

α1-Antitrypsin (A1AT), a highly polymorphic 52-kDa glycoprotein with at least 100 alleles, functions as the major inhibitor of neutrophil elastase (1) . Two allelic variants, PiZ and PiS , frequently lead to A1AT deficiency, which can manifest clinically as emphysema and, less frequently, as liver disease in neonates (2) . Recently, A1AT deficiency has been recognized to play a role in the development of bronchial asthma (3) . The inflammatory process in bronchial asthma involves various mechanisms by which the inflammatory response is perpetuated. These include, for example, macrophage-mediated or mast cell-mediated recruitment and activation of inflammatory cells as well as amplification of neutrophil recruitment and activation. The activated neutrophils release neutrophil elastase, which then can stimulate production of cytokines (4) . For the deficiency variants of A1AT, PiZ and PiS , reduced elastase inactivation capacity has been demonstrated. A diminished inhibitory activity has also been described for PiM2 and PiM1(ala) (5) . The resulting high elastase activity will stimulate cytokine production and can further amplify recruitment of activated neutrophils in the inflamed airway (3)(6)(7) . Recently, an association of the variants PiM2 and PiM1(ala) with bronchial asthma has been reported (5)(8)(9) . Thus, the measurement of the serum A1AT and the identification of the A1AT phenotype are clinically important. Traditionally, the A1AT phenotype has been analyzed by isoelectric focusing. This analysis is tedious and prone to difficulties in interpretation (10) . PiZ and PiS genotypes may be determined by DNA-based methods such as restriction fragment length polymorphism, allele-specific oligonucleotide hybridization, allele-specific amplification, direct sequencing, dual-color detection by ligase-mediated analysis, temperature or denaturing gradient gel electrophoresis, and PCR-mediated site-directed mutagenesis (11)(12)(13)(14)(15)(16) …

Prognostic value of serum alpha-1-antitrypsin in hepatocellular carcinoma

To evaluate serum alpha-1-antitrypsin (A1AT) as a prognostic factor in hepatocellular carcinoma, we studied 75 consecutive patients (60 male, 15 female, mean age ± SD 63.0 ± 9.3 years) in whom hepatocellular carcinoma developed with pre-existing cirrhosis. Median survival time was 245 days (range 4–1568+). 30 patients had serum A1AT concentration of ⩽2.20 g/1 (Group A) while 45 (Group B) had alpha-1-antitrypsin > 2.20 g/1. Median survival was 518 days in Group A and 81 days in Group B (Mantel-Cox 20.95, P < 0.0001; hazard ratio 0.26, 95% confidence limits 0.15–0.46). By stepwise survival analysis, alpha-1-antitrypsin together with bilirubin, tumour size and blood urea nitrogen were chosen among 17 variables as the only independent predictors of survival. We conclude that measurement of serum A1AT concentration might be useful as an inexpensive, widely available prognostic marker of hepatocellular carcinoma.

Polymerase chain reaction-mediated site-directed mutagenesis detection of Z and S alpha-1-antitrypsin alleles in family members

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder with a reduction in serum A1AT levels. In a large family, we used a polymerase chain reaction (PCR)-mediated, site-directed mutagenesis assay to detect the two most common A1AT deficient variants, Z and S. By coamplification, using primers for both the Z and S mutations, we were able to detect heterozygous and homozygous genotypes for both mutations in a single reaction. We compared our results with phenotype studies obtained by standard immunofixation and isoelectric focusing techniques at two reference laboratories. Whereas PCR and isoelectric focusing agreed completely, there were five discrepancies in the results obtained by the immunofixation procedure. The reference laboratory that provided these discrepant results later informed us of a quality control problem that accounted for their error. The family study included 12 individuals representing three generations. Two individuals were MM homozygotes, three were MZ heterozygotes, four were MS heterozygotes, and three were SZ heterozygotes. A thirteenth family member was diagnosed as a ZZ homozygote at another institution. We have shown that this PCR coamplification technique provides accurate information about the M, S, and Z alleles that is at least as useful as current reference laboratory methodologies.

Acute phase proteins in serum and cerebrospinal fluid in the course of bacterial meningitis

We carried out estimations of the following acute phase proteins: C-reactive protein (CRP), alpha-l-antitrypsin (AAT), alpha-1-acid glycoprotein (AAG), alpha-2-ceruloplasmin (CER), and alpha-2-haptoglobin (HPT) in serum and in cerebrospinal fluid (CSF) in patients with bacterial meningitis (BM, n = 30) and viral meningitis (VM, n = 30). We have shown that determinations of concentrations of AAG and CRP in serum and CER in CSF are useful in differentiation between BM and VM. The diagnostic power of these three tests (the areas under their ROC curves equal 0.942, 0.929, and 0.931, respectively) is bigger, though statistically not significantly, than that of traditional parameters of BM in CSF, i.e., total protein concentration and white blood cell count. Determination of AAG, CRP, and AAT in serum is a valuable monitoring marker in the course of BM treatment. Convenience of serum sampling constitutes an advantage over traditional BM parameters in CSF.

Alpha1-Antitrypsin in Tropical Pulmonary Eosinophilie

Serum alpha-antitrypsin (A1AT) was measured in 36 patients (group A) with tropical pulmonary eosinophilia (TPE) and 30 patients with pulmonary eosinophilia with associated worm infestation (group B). Serum A1AT level was also measured in 42 healthy subjects (group C). In 28 patients (group A, 15 and group B, 13), the serum A1AT level was below 60 percent of the mean A1AT value of the control group. The A1AT-deficient patients had a more severe form of illness with recurrence and relapse, and they were generally refractory to treatment. In these deficient patients, a significant negative correlation (p < 0.02) between A1AT and absolute blood eosinophil counts (AEC) was observed. In 21 patients (group A, B and group B, 13), the effect of therapy with either diethylcarbamazine or deworming or both was observed. This showed a significant posttreatment rise of A1AT level with a significant fall in AEC (p < 0.02) as compared to their respective pretreatment levels. Serum A1AT levels in either or both parents of 11 patients (group A, 5 and group B, 6) were found to be within normal levels. The significant rise in A1AT levels with a significant fall in AEC levels in the patients following therapy and the absence of A1AT deficiency in the parents examined indicate that the A1AT deficiency observed in TPE is probably acquired.

Clinical usefulness of alpha-1-antitrypsin in the diagnosis of hepatocellular carcinoma.

Serum levels of alpha-1-Antitrypsin(AAT) were determined in 42 patients with hepatocellular carcinoma(HCC), 5 patients with metastatic liver cancer from stomach adenocarcinoma, 10 patients with liver cirrhosis, 10 patients with chronic hepatitis, and 66 controls by rocket immunoelectrophoresis using rabbit antiserum. The mean level of serum AAT was 225.5 +/- 73.0 mg/dl in 66 controls. The serum AAT in patients with HCC was 428.7 +/- 123.3 mg/dl, which was significantly higher than those in the controls and in patients with liver cirrhosis or chronic hepatitis(p less than 0.02). The level of AAT in metastatic liver cancer was similar to that in HCC. The positive cut-off value for elevation of serum AAT in this study was determined as above 445 mg/dl, the mean plus 3 standard deviations in the controls. Elevations of serum AAT were observed in 54.8%, 60.0%, and 10.0% of patients with HCC, metastatic liver cancer, and liver cirrhosis, respectively, while none of the patients with chronic hepatitis or the controls was positive. The serum AAT levels in 42 patients with HCC were analyzed with regard to sex, age, serum albumin, HBsAg, alpha-fetoprotein(AFP), and diameter of HCC, with no significant differences being observed between these factors and the serum AAT levels except for the diameter of the HCC. The positive rate in the HCC with a diameter of 10 cm or more was 74.1%, which was a significantly higher rate compared with 20.0% in the HCC with diameters less than 10cm. The positive rate of AFP for HCC was 61.9%, when 500 ng/ml of AFP was used as the cut-off value.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha1‐antitrypsin and survival in pancreatic cancer

The association between serum levels of alpha 1-antitrypsin (a1AT) at the time of diagnosis and survival was studied in a group of 44 patients with confirmed pancreas cancer. All 44 patients were followed until time of death, which occurred in all cases from pancreas cancer, with a median time of 3 months and a range of 0.5 to 16 months. Cox's proportional hazards model was utilized in the analysis controlling for sex, age and tobacco smoking. Males, older patients and smokers have higher fatality rates, but none of these relations was statistically significant. By contrast, there was a statistically highly significant association of increased levels of serum a1AT, at the time of diagnosis of the cancer of pancreas, with shorter survival; patients with serum a1AT higher by 100mg/100ml had a 57% higher fatality rate. These results indicate that serum a1AT represents a clinically interesting prognostic factor in pancreas cancer.

ROC Analysis of Diagnostic Performance Utilizing Serum Antiproteases in Cancer Patients

Assessment of the accuracy of diagnostic procedures is made independent of diagnostic criteria by means of a receiver-operating-characteristics (ROC) curve. We performed ROC analysis for the major serum antiproteases: alpha-1-antitrypsin (A1AT) and alpha-2-macroglobulin (A2M), in 99 cancer patients compared with 71 normal individuals. A1AT and A2M were significantly higher in cancer patients (p less than 0.0005). By comparing true positive and false positive rates for different serum levels, ROC analysis showed that serum A1AT quantification seems more useful in clinical practice than serum A2M.

Alpha-1-antitrypsin augmentation therapy for alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin (A1AT) deficiency is a genetic disorder characterized by low serum levels of A1AT and a high risk for the development of emphysema. A1AT is the principal inhibitor of neutrophil elastase, such that a deficiency of AIAT results in insufficient anti-elastase protection in the lower respiratory tract, thus allowing neutrophil elastase to destroy alveolar structures. The goal of A1AT augmentation therapy in A1AT deficiency is to raise lung A1AT levels and anti-neutrophil elastase capacity to levels that will provide adequate protection against neutrophil elastase, thereby preventing the lung from further elastase-mediated degradation. Studies with intravenous administration of human A1AT (60 mg/kg at weekly intervals) demonstrate that serum A1AT levels increased from an average 33 ± 8 mg/dl pre-infusion to a steady-state trough level of 117 ± 4 mg/dl, well above the projected threshold protective serum level of A1AT. The infused A1AT diffused into the lung and significantly augmented the epithelial lining fluid A1AT levels, rising from an average 0.44 ± 0.16 μM (pre-infusion) to 2.62 ± 1.29 μM at the nadir level just prior to the next infusion. Of critical importance is the fact that the A1AT that diffused into the lung was active as an inhibitor or neutrophil elastase, resulting in significant augmentation of epithelial lining fluid anti-neutrophil elastase capacity and normalization of the lung anti-elastase protection. In the over 800 weekly infusions administered, no significant adverse reactions have occurred. These findings demonstrate that long-term augmentation therapy with weekly infusions of A1AT is a rational, safe, and biochemically effective therapy for A1AT deficiency. Alpha-1-antitrypsin (A1AT) deficiency is a genetic disorder characterized by low serum levels of A1AT and a high risk for the development of emphysema. A1AT is the principal inhibitor of neutrophil elastase, such that a deficiency of AIAT results in insufficient anti-elastase protection in the lower respiratory tract, thus allowing neutrophil elastase to destroy alveolar structures. The goal of A1AT augmentation therapy in A1AT deficiency is to raise lung A1AT levels and anti-neutrophil elastase capacity to levels that will provide adequate protection against neutrophil elastase, thereby preventing the lung from further elastase-mediated degradation. Studies with intravenous administration of human A1AT (60 mg/kg at weekly intervals) demonstrate that serum A1AT levels increased from an average 33 ± 8 mg/dl pre-infusion to a steady-state trough level of 117 ± 4 mg/dl, well above the projected threshold protective serum level of A1AT. The infused A1AT diffused into the lung and significantly augmented the epithelial lining fluid A1AT levels, rising from an average 0.44 ± 0.16 μM (pre-infusion) to 2.62 ± 1.29 μM at the nadir level just prior to the next infusion. Of critical importance is the fact that the A1AT that diffused into the lung was active as an inhibitor or neutrophil elastase, resulting in significant augmentation of epithelial lining fluid anti-neutrophil elastase capacity and normalization of the lung anti-elastase protection. In the over 800 weekly infusions administered, no significant adverse reactions have occurred. These findings demonstrate that long-term augmentation therapy with weekly infusions of A1AT is a rational, safe, and biochemically effective therapy for A1AT deficiency.

Alpha-1-antitrypsin augmentation therapy for alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin (A1AT) deficiency is a genetic disorder characterized by low serum levels of A1AT and a high risk for the development of emphysema. A1AT is the principal inhibitor of neutrophil elastase, such that a deficiency of A1AT results in insufficient anti-elastase protection in the lower respiratory tract, thus allowing neutrophil elastase to destroy alveolar structures. The goal of A1AT augmentation therapy in A1AT deficiency is to raise lung A1AT levels and anti-neutrophil elastase capacity to levels that will provide adequate protection against neutrophil elastase, thereby preventing the lung from further elastase-mediated degradation. Studies with intravenous administration of human A1AT (60 mg/kg at weekly intervals) demonstrate that serum A1AT levels increased from an average 33 ± 8 mg/dl pre-infusion to a steady-state trough level of 117 ± 4 mg/dl, well above the projected threshold protective serum level of A1AT. The infused A1AT diffused into the lung and significantly augmented the epithelial lining fluid A1AT levels, rising from an average 0.44 ± 0.16 μM (pre-infusion) to 2.62 ± 1.29 μM at the nadir level just prior to the next infusion. Of critical importance is the fact that the A1AT that diffused into the lung was active as an inhibitor or neutrophil elastase, resulting in significant augmentation of epithelial lining fluid anti-neutrophil elastase capacity and normalization of the lung anti-elastase protection. In the over 800 weekly infusions administered, no significant adverse reactions have occurred. These findings demonstrate that long-term augmentation therapy with weekly infusions of A1AT is a rational, safe, and biochemically effective therapy for A1AT deficiency.

Molecular basis of alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder associated with a major reduction in serum A1AT levels. Clinically, A1AT deficiency is associated with emphysema in adults and, less commonly, liver disease in neonates. A1AT is a 52-kDa, 394-amino acid, single-chain glycoprotein normally present in serum at 150 to 350 mg/dl. The A1AT gene, composed of seven exons dispersed over 12 kb of chromosomal segment 14q31-32.3, is expressed in heptocytes and mononuclear phagocytes. The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), is a globular molecule composed of nine alpha-helices and three beta-pleated sheets. The major function of A1AT is to inhibit neutrophil elastase; A1AT does so through an active site centered around Met358 contained within an external stressed loop on the surface of the molecule. A1AT is a highly pleomorphic protein with greater than 75 variants determined at the protein and/or gene level. These variants can be categorized into four groups according to their serum A1AT level and function: normal, deficient, dysfunctional, and absent. There are two important salt bridges within the A1AT molecule (Glu342—Lys290; Glu263—Lys367); a mutation in the A1AT gene causing disruption of either salt bridge causes distinct molecular pathology resulting in reduced serum A1AT levels. Clinically relevant variants can be distinguished by a combination of isoelectric focusing of serum, restriction fragment length analysis of genomic DNA, oligonucleotide probes, and direct sequencing of the variant A1AT genes. Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder associated with a major reduction in serum A1AT levels. Clinically, A1AT deficiency is associated with emphysema in adults and, less commonly, liver disease in neonates. A1AT is a 52-kDa, 394-amino acid, single-chain glycoprotein normally present in serum at 150 to 350 mg/dl. The A1AT gene, composed of seven exons dispersed over 12 kb of chromosomal segment 14q31-32.3, is expressed in heptocytes and mononuclear phagocytes. The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), is a globular molecule composed of nine alpha-helices and three beta-pleated sheets. The major function of A1AT is to inhibit neutrophil elastase; A1AT does so through an active site centered around Met358 contained within an external stressed loop on the surface of the molecule. A1AT is a highly pleomorphic protein with greater than 75 variants determined at the protein and/or gene level. These variants can be categorized into four groups according to their serum A1AT level and function: normal, deficient, dysfunctional, and absent. There are two important salt bridges within the A1AT molecule (Glu342—Lys290; Glu263—Lys367); a mutation in the A1AT gene causing disruption of either salt bridge causes distinct molecular pathology resulting in reduced serum A1AT levels. Clinically relevant variants can be distinguished by a combination of isoelectric focusing of serum, restriction fragment length analysis of genomic DNA, oligonucleotide probes, and direct sequencing of the variant A1AT genes.

Molecular basis of alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin (A1AT) deficiency is an autosomal hereditary disorder associated with a major reduction in serum A1AT levels. Clinically, A1AT deficiency is associated with emphysema in adults and, less commonly, liver disease in neonates. A1AT is a 52-kDa, 394-amino acid, single-chain glycoprotein normally present in serum at 150 to 350 mg/dl. The A1AT gene, composed of seven exons dispersed over 12 kb of chromosomal segment 14q31–32.3, is expressed in heptocytes and mononuclear phagocytes. The A1AT protein, a member of the class of protease inhibitor proteins known as serpins (serine protease inhibitors), is a globular molecule composed of nine alpha-helices and three beta-pleated sheets. The major function of A1AT is to inhibit neutrophil elastase; A1AT does so through an active site centered around Met 358 contained within an external stressed loop on the surface of the molecule. A1AT is a highly pleomorphic protein with greater than 75 variants determined at the protein and/or gene level. These variants can be categorized into four groups according to their serum A1AT level and function: normal, deficient, dysfunctional, and absent. There are two important salt bridges within the A1AT molecule (Glu 342—Lys 290; Glu 263—Lys 387); a mutation in the A1AT gene causing disruption of either salt bridge causes distinct molecular pathology resulting in reduced serum A1AT levels. Clinically relevant variants can be distinguished by a combination of isoelectric focusing of serum, restriction fragment length analysis of genomic DNA, oligonucleotide probes, and direct sequencing of the variant A1AT genes.

The reactivity of alpha-1-antitrypsin with Lens culinaris agglutinin and its usefulness in the diagnosis of neoplastic diseases of the liver.

The reactivity of alpha-1-antitrypsin (AAT) with Lens culinaris agglutinin (LCA) was studied by crossed immuno-affinity electrophoresis of the sera of 246 subjects from 6 groups (acute virus hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma (HCC), carcinoma metastatic to the liver and normal controls). Two species of AAT (LCA-reactive and -nonreactive species) were detected on crossed immuno-affinity electrophoresis in a gel containing LCA. The percentages of LCA-reactive species of AAT in neoplastic diseases of the liver were significantly higher than those in benign liver diseases and normal controls. There was no correlation between the percentage of LCA-reactive species of AAT and serum AAT concentration in any group. Furthermore, in studying 15 pairs of serum samples before and after the subsequent development of HCC, the percentage of LCA-reactive species of AAT after HCC occurrence was significantly higher than that before, although there was no statistically significant difference between the serum AAT concentration before and after development of the disease. The latter 15 patients were all of the normal protease inhibitor phenotype (PiMM) and no change in phenotype was observed before and after the development of HCC. The results indicate that measurement of the reactivity of AAT with LCA can be a useful marker for the diagnosis of HCC and carcinoma metastatic to the liver, especially when serum concentrations of alpha-foetoprotein or other tumour markers are within the normal ranges.

Detection of alpha-1-antitrypsin in hepatocytes in acute and chronic hepatitis

Twelve of sixteen consecutive needle biopsies of liver with either acute or chronic hepatitis showed positive immunohistochemical staining for alpha-1-antitrypsin (AAT). Only two of the positive biopsies contained numerous, large periodic acid-Schiff positive, diastase resistant (PAS-D) globules in periportal hepatocytes; both patients were Pi MZ but only one had a low serum AAT concentration. The other 15 patients had normal or elevated serum AAT. The accumulation of AAT in hepatocytes, demonstrated by sensitive immunohistochemical staining, may indicate increased synthesis and/or impaired secretion of AAT occurring in association with various types of hepatitis.

Alpha-1-antitrypsin in human macrophages.

Preliminary studies have suggested that alpha-1-antitrypsin (A1AT) is a useful immunohistochemical marker of histiocytes (monocytes/macrophages) and malignant tumours derived from them. To confirm the reliability of this marker a wide variety of benign and malignant lymphoreticular cells and tissues have been stained by the immunoperoxidase technique for A1AT and positive staining was found to be confined to histiocytes. Immunodiffusion, isotope labelling, and isoelectric focusing studies performed on cell lysates confirmed that the positive staining shown by monocytes and malignant histiocytes is due to the presence of A1AT identical with serum A1AT and that this material is synthesised by these cells rather than taken up from their environment. Positive immunoperoxidase staining for A1AT is thus a reliable marker of lymphoreticular neoplasms of true histiocytic origin.

558 GASTROINTESTINAL CLEARANCE OF ALPHA-1-ANTITRYPSIN: A METHOD FOR DIAGNOSING PROTEIN-LOSING ENTEROPATHY

Because of the unavailability of alternate methods to determine protein-loss, we measured the gastrointestinal clearance of alpha-1-antitrypsin (AAT), by a modification of a European technique. We studied eight patients, three with hypoalbuminemia and disorders suggestive of protein-losing enteropathy, and five with or without hypoalbuminemia and gastrointestinal disorders not necessarily associated with protein loss. Clearance of AAT was calculated from the following equation: CAAT = (FxV)/P where C is in cc/days; F = fecal concentration of AAT in cc/1; V = fecal volume in cc/day; P = serum AAT in mg/1. Stools were collected over a three day period and serum AAT obtained on each of three days and averaged in some patients, and from two or one specimen in other patients. The stool was homogenized, and a one gram aliquot resuspended in distilled water. AAT was measured by radioimmuno-diffusion using standard techniques. Serum AAT was measured by standard techniques.We conclude that a three day stool collection with determination of CAAT may be useful in identifying children with protein losing enteropathy.

Antiprotease activity in amniotic fluid in a family with a1 antitrypsin deficiency (a1-AT)

We studied a family in which a 21/2 years old child exhibited liver cirrhosis in association with a1-AT deficiency of the PiZZ genotype. The diagnosis was established in the proband at the time her mother was again pregnant and in her 8th month of gestation. Antiprotease activity was measured by the trypsin inhibitory method of Eriksson and by immunodiffusion techniques. Amniotic fluid was obtained from the PiZ heterozygote prior to delivery by needle puncture of the amniotic membrane. This fluid was contrasted with term amniotic fluid from 5 PiMM women and 5 fluids obtained during the l6-l8th weeks of gestation for diagnosis of other genetic diseases. The term control specimens had a trypsin inhibitory capacity = 0.031-0.106 mg/ml, first trimester fluids = 0.073-0.175 mg/ml. Immunodiffusion indicated that serum a1-AT was present in amniotic fluid, concentration = 0.293 ± 0.078 mg/ml. The child from the pregnancy under study had hepatic disease and was a PiZZ homozygote. Her amniotic fluid had antitrypsin activity 10% of controls (0.006 mg/ml). The feasibility of antenatal diagnosis is suggested.