Genetic typing of alpha1-antitrypsin by immunofixation electrophoresis, identification of subtypes of Pi M.

Prolonged electrophoresis in alkaline agarose gels, followed by immunofixation, is a valuable addition to acid starch gel electrophoresis and isoelectric focusing for the genetic phenotyping of alpha1-antitrypsin. This technique is helpful in clarifying certain variants and in ascertaining types in serum and amniotic fluid samples with secondary changes. In addition, heterogeneity may be detected within the variants found at pH 4.95, analogous to hemoglobin polymorphism. Two "new" variants, PiMLamb and PiMBaldwin, have been detected by a combination of immunofixation electrophoresis and acid starch gel electrophoresis.     

N-[(carbamoylmethyl)amino]ethanesulfonic acid improves phenotyping of alpha 1-antitrypsin by isoelectric focusing on agarose gel

The hereditary deficiency variants of alpha 1-antitrypsin that are associated with diseases such as emphysema are usually identified by use of isoelectric focusing on polyacrylamide gels. Agarose is a simpler, faster, safer, and more reliable medium for this, but resolution often is not as good. I describe a method in which the ultrathin agarose gel contains N-[(carbamoylmethyl)amino]ethanesulfonic acid as a "separator," to flatten the pH gradient and improve separation of the alpha 1-antitrypsin isoforms. The resolution obtained equals or surpasses that of conventional methods based on use of either polyacrylamide or agarose. Haptoglobin, which interferes with isoelectric focusing on polyacrylamide, does not interfere with this method; other advantages are also discussed.     

Isoelectric focusing in agarose: classification of genetic variants of alpha 1-antitrypsin

Polyacrylamide has been the matrix of choice for isoelectric focusing owing to the virtual absence of electroendosmosis in this medium. Certain inherent limitations associated with polyacrylamide have prompted some investigators to use low-electroendosmosis agarose for isoelectric focusing, but with limited success thus far. We have developed a method for isoelectric focusing in agarose for the classification of alpha 1-antitrypsin variants. Sera are applied directly to agarose gels containing a pH 4-5 ampholyte mixture, focused for less than 1 h, and directly immunofixed. Resolution of major bands is equivalent to polyacrylamide, and Pi M subtypes can be distinguished without the use of a separator. This application demonstrates the high resolution of isoelectric focusing in agarose, a more practical and convenient matrix than polyacrylamide.     

Evaluation of a new Sebia isoelectrofocusing kit for alpha 1-antitrypsin phenotyping with the Hydrasys System.

BACKGROUND: Laboratory evaluation of alpha 1-antitrypsin (A1AT) deficiency is generally performed by determination of A1AT concentrations and identification of specific allelic variants by phenotyping. For this purpose, we evaluated a new Hydragel 18 A1AT Isofocusing kit on the semi-automatic Hydrasys System (Sebia) for the determination of A1AT phenotypes by isoelectrofocusing on ready-to-use agarose gels with specific immunological detection. METHODS: Serum samples from 66 patients were analysed with this new kit in comparison with the conventional and manually performed isoelectrofocusing method on polyacrylamide gels with Coomassie Blue staining. RESULTS: A1AT phenotypes showed comparable iso-electrofocusing patterns in both systems. The good within-gel reproducibility of this kit was demonstrated using two normal serum samples (M1 and M1M2 phenotypes) and six pathological serum samples with different phenotypes (MS, SS, SZ, MZ, ZZ). A sensitivity study was undertaken by performing serial dilutions on a serum with a ZZ phenotype containing 0.27 g/L A1AT. The detection limit was 0.050 g/L. CONCLUSIONS: This new method is highly specific, rapid and simple to perform. It improves identification of not only the most common but also various rare A1AT phenotypes. It appears to be suitable for routine analysis and screening applications in a clinical laboratory setting.     

Improved method for identifying alpha 1-antitrypsin Pi M subtypes by isoelectric focusing in agarose

We describe an improved method for the classification of alpha 1-antitrypsin variants by isoelectric focusing in agarose. Identification of the three Pi M subtypes can now be made by using a narrow-range carrier ampholyte (pH 4.2-4.9) and pretreating serum with dithioerythritol-iodoacetic acid to enhance band resolution. Phenotype results for two groups of Pi M homo- and heterozygotes are compared to illustrate the improved accuracy of the new method.     

Leucocyte-associated plasma proteins. Association of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, transferrin and haptoglobin with human lymphocytes, monocytes, granulocytes and a promyelocytic leukaemic cell line (HL-60)

The distribution of prealbumin, albumin, orosomucoid, alpha 1-antitrypsin, haptoglobin and transferrin, including their electrophoretic heterogeneous variants, was studied in isolated lymphocytes, monocytes and granulocytes and in a human promyelocytic cell line (HL-60) by crossed immunoelectrophoresis. Prealbumin, albumin and transferrin were present in lymphocytes, monocytes and granulocytes, whereas the cellular variants of orosomucoid and haptoglobin were present only in granulocytes. alpha 1-Antitrypsin was present in four electrophoretic variants which were differently distributed among the various cell types. Synthesis of alpha 1-antitrypsin by monocytes, granulocytes and HL-60 cells was demonstrated by 14C-leucine incorporation. The six plasma proteins could not be removed from intact cells by incubation with the respective antibodies at 0 degrees C, or iodinated by lactoperoxidase catalysed iodination at 23 degrees C. They were, however, readily solubilized by freeze-lysis of the cells, suggesting an intracellular localization. Compared to their plasma counterparts none of the proteins differed in their hydrophobic properties but the carbohydrate residues of orosomucoid, alpha 1-antitrypsin and haptoglobin were different. The pattern of disappearance of the proteins from the cells during incubation suggested that the localization of albumin and transferrin in relation to the cells differed from that of the other proteins.     

Optimal conditions for protease use in the assay of serum mitochondrial aspartate aminotransferase

The optimal conditions for selective proteolytic inactivation of cytosolic aspartate aminotransferase (c-AST) to determine mitochondrial aspartate aminotransferase (m-AST) in serum were studied. Protease 401 was found to be effective over a pH range of 6.0-10.0. A pH of 9.5 with 0.5% albumin in the reagent mixture was determined to be optimal for inactivation of c-AST and preservation of m-AST, lactic dehydrogenase (LDH), and malic dehydrogenase (MDH) in the assay procedure. The presence of serum endogenous protein inhibitors such as alpha 1-antitrypsin and alpha 2-macroglobin did not inhibit protease 401.     

Simplified alpha1-antitrypsin phenotyping by immunofixation of acid-starch gels.

Immunofixation of acid-starch gels represents a simplified method for alpha1-antitrypsin phenotyping. This technique reduces the amount of work and time involved in phenotyping by eliminating the need for crossed-immunoelectrophoresis. Its major purpose is to directly enhance the staining quality of the minor bands, especially the Z bands on the acid-starch gel. It is hoped that this modified method will allow more wide-spread use of antitrypsin phenotyping, especially in the routine clinical chemistry laboratory.     

Silver-stained phenotyping of alpha 1-antitrypsin in dried blood and serum specimens

In this technique for determining the electrophoretic phenotype of alpha 1-antitrypsin in dried blood or serum specimens, the adsorbed material is eluted with a concentrated solution of dithiothreitol, focused on polyacrylamide thin-layer gel, and made visible with silver stain. With this staining technique all normal and pathological alpha 1-antitrypsin phenotypes can be detected. The procedure is relatively simple, inexpensive, and suitable for use in large-scale screening for alpha 1-antitrypsin deficiency in selected populations.     

Catabolic rate of alpha1-antitrypsin of Pi type M and Z in man.

1. Human alpha1-antitrypsin was isolated from three Pi M and two Pi Z subjects without alteration of its microheterogeneity. The purified proteins were labelled with either 125I or 131I by a lactoperoxidase method. 2. The disappearance rate of two types of alpha1-antitrypsin were studied after simultaneous injection of labelled M-protein and Z-protein into Pi M subjects. 3. The ratio of extravascular to plasma pools of alpha1-antitrypsin ranged between 1-2 and 1-6 with no difference between M- and Z-protein. The mean fractional catabolic rates of M-protein and Z-protein were respectively 0-26 and 0-40 per day. 4. The difference in catabolic rate of Z- and of M-protein is too small to explain why the alpha1-antitrypsin content of the blood in Pi ZZ subjects is only 15% of that normally found in Pi MM subjects. The low alpha1-antitrypsin in Pi ZZ subjects appears mainly to be due to a low rate of biosynthesis.     

Catabolic rate of alpha1-antitrypsin of of Pi types S, and MMalton and of asialylated M-protein in man.

1. Human alpha1-antitrypsin was isolated with preserved microheterogeneity from subjects of Pi types M, S and MMalton. The M-protein was partially (20%) and completely desialylated. The proteins were labelled with either 125I or 131I. 2. The disappearance rate of these alpha1-antitrypsins was studied after simultaneous injection of the two types of labelled protein into Pi M subjects. The fractional catabolic rates of S- and MMalton-protein were 0.36 and 0.34 day(-1) respectively compared with 0.28 day(-1) for M-protein. The ratio of extravascular to plasma pools was 1.4 for S- protein and 1.6 for MMalton-protein. The 20% desialylated M-protein showed an increase of about 100% in its fractional catabolic rate. The disappearance rate of completely desialylated alpha1-antitrypsin was extremely rapid. 3. The slightly higher fractional catabolic rate of S- than of M-protein can only partly explain the 40% lower plasma concentration in subjects of Pi type S. Similarly the slight increase in catabolic rate of Pi MMalton- protein is too small to explain why the alpha1-antitrypsin content of the blood in Pi MMalton subjects is only 15% of that normally found. A low hepatic secretion seems to be the major cause of the low alpha1-antitrypsin concentration found in subjects of types Pi S and MMalton, as in Pi type Z.     

Variability of plasma proteins according to molecular size. Long-term and short-term intra-individual variation

The intra-individual variations in serum concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin were determined using high precision analytical methods. The long-term (3 months) variations were 8.2% for alpha 1-antitrypsin and 2.9% for alpha 2-macroglobulin in five males and five females. The coefficients of variation for albumin were 1.5 and 3.4% for males and females, respectively. In males the long-term variations of albumin and alpha 2-macroglobulin were highly correlated. The short-term (2 days) intra-individual variations in six males were 2.5, 3.8 and 3.4% for alpha 1-antitrypsin, albumin and alpha 2-macroglobulin respectively (coefficients of variation). A diurnal variation was found for albumin with maximal concentrations at 18.00 hours. At 6.00 and 10.00 hours the fractional concentrations of alpha 1-antitrypsin and albumin were lower than for alpha 2-macroglobulin. The variations of the three proteins were positively correlated.     

Immunohistochemical studies in the differential diagnosis of malignant fibrous histiocytoma

Malignant fibrous histiocytomas (MFH) belong to the most frequent soft tissue tumours in adults and have to be discriminated from other tumours with similar morphology. Various tumour markers aid the differential diagnosis. Twenty cases of MFH were studied immunohistochemically using antibodies to vimentin, TPA, desmin, lysozyme, alpha 1-antitrypsin, alpha 1-antichymotrypsin, S-100 protein, neurone-specific enolase (NSE), laminin, fibronectin and ferritin. Vimentin and lysozyme were found in the tumour cells of all, alpha 1-antitrypsin of 18, alpha 1-antichymotrypsin of 19, fibronectin of 16 and ferritin of 12 cases. Antibodies of TPA, desmin, S-100 protein, NSE and laminin did not reveal positive immunoreactivity. Exclusion of spindle-cell carcinoma can be made by positive vimentin and negative TPA reactivity, of melanoma by negative S-100 reactivity, and of leio- and rhabdomyosarcoma by lack of desmin immunoreactivity. Schwannomas contain S-100 protein, but lack lysozyme, alpha 1-antitrypsin, alpha 1-antichymotrypsin and fibronectin. Pleomorphic liposarcomas cannot be distinguished from MFH on the basis of immunohistochemical staining. Vimentin, alpha 1-antitrypsin, alpha 1-antichymotrypsin and fibronectin can, therefore, be regarded as useful markers in the differential diagnosis of MFH.     

Typing of genetic variants of alpha 1-antitrypsin from dried blood

The alpha 1-antitrypsin phenotype was determined from both plasma and dried blood in 35 children and adolescents who had or were suspected to have alpha 1-antitrypsin deficiency. A disc of paper with dried blood was eluted and analysed by flat bed electrofocusing on polyacrylamide gel. The gel was stained according to a silver-staining technique until the microheterogeneous bands appeared with adequate intensity. The alpha 1-antitrypsin patterns of plasma and dried blood were identical. The stability of the dried blood samples was 3 days at room temperature, up to 1 week at +4 degrees C, and up to 1 month at -20 degrees C. The alpha 1-antitrypsin screening method and the Pi-typing procedure described in this study may be combined in future screening studies using dried blood specimens obtained for the Guthrie test.     

Synthesis and secretion of α2-macroglobulin by human hepatocytes in culture

Hepatocytes were isolated by application of the two-step collagenase perfusion technique to pieces of human liver. The cells were incubated in serum-free medium or 10% FCS-medium supplemented with insulin, glucagon and dexamethasone, and kept in culture for more than 2 weeks. Seventy-five per cent of the medium was changed regularly and assayed for alpha 2-macroglobulin (alpha 2-M), pregnancy zone protein, alpha 1-antitrypsin and albumin by means of ELISA. Significant amounts of alpha 2-macroglobulin were present in all cultures. During incubation, alpha 2-M accumulated in the medium and the quantity of alpha 2-M released from the cells by far exceeded protein associated with hepatocytes prior to incubation. In 24 h 10(6) hepatocytes secreted 160.5 +/- 82.2 ng of alpha 2-M (mean +/- SD, n = 5). Cell-associated, as well as secreted alpha 2-M appeared to be on native form, as determined by immunoisolates from lysed cells and culture supernatants. Pregnancy zone protein was only detected in about 50% of the cultures and its rate of secretion was less than 2 ng 24 h-1 per 10(6) cells. In contrast, culture medium contained considerable quantities of alpha 1-antitrypsin and albumin. In 24 h, 10(6) hepatocytes released greater than 2 micrograms alpha 1-antitrypsin and greater than 5 micrograms albumin. The present study suggests the hepatocyte to be of major importance for the synthesis of intravascular alpha 2-M.     

Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants

We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb K?ln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb K?ln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.     

Immunohistochemical demonstration of alpha 1-antichymotrypsin and alpha 1-antitrypsin in salivary gland pleomorphic adenomas of children.

Twenty-five benign pleomorphic adenomas of salivary glands in children were studied with immunohistochemical techniques in order to characterize the cell types comprising the epithelial and so-called "mesenchymal" regions of the tumors. The antisera against alpha 1-antichymotrypsin (alpha 1-ACT) and alpha 1-antitrypsin (alpha 1-AT) were used to stain in normal salivary gland tissue as well as in pleomorphic adenoma. In normal salivary glands, alpha 1-ACT was localized to the intercalated duct and serous acinar cells. On the other hand, there was positive staining for alpha 1-AT in the intercalated and striated duct cells. Twenty-five cases (100%) of pleomorphic adenomas in children displayed positivity to alpha 1-ACT staining and 22 cases (88%) showed a positive staining for alpha 1-AT. alpha 1-ACT staining was particularly intense in chondrocyte-like cells of 20 cases (80%), in inner tubular cells of 16 (64%) and cyst-lining cells of 12 (52%). The limited number of tumor cells which were called plasmatoid or hyaline cells and squamous epithelial cells, were positive for alpha 1-ACT. None of the outer tubular cells and hyalinous material was positively stained for alpha 1-ACT. A strong positive reaction for alpha 1-AT was observed in chondrocyte-like cells of 15 cases (60%). Inner tubular cells were positive for alpha 1-AT in 12 cases (48%), plasmatoid or hyaline cells in 10 (40%) and cyst-lining cells in 8 (35%). Squamous epithelial cells, clear cells, secretory product and hyalinous material were positive for alpha 1-AT in some cases. Chondroid matrix and myxoid stroma had no reaction with both antibodies. The biological role of alpha 1-ACT and alpha 1-AT with a wide immunohistochemical distribution in pleomorphic adenomas of children may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

Effects of posture on concentrations of serum proteins in healthy adults. Dependence on the molecular size of proteins

The effect of posture on changes in protein composition of serum and its dependence on molecular size has been investigated in forty individuals classified by age and sex. Blood specimens were drawn (A) at 0700 hours with persons still resting in bed after an overnight sleep; (B) at 0815 hours, in sitting position after 1 h of moderate exercise and again (C) at 0915 hours, lying in bed after 1 h of recumbency. Concentrations of alpha 1-antitrypsin, albumin and alpha 2-macroglobulin in serum were determined with high analytical precision. The mean fractional increases of concentrations from lying (A) to sitting (B) were 0.069 for alpha 1-antitrypsin and 0.063 for albumin, but lower, 0.040, for alpha 2-macroglobulin. Thus these increases, being related to molecular size, were not simply caused by posture induced changes of plasma volume. Considering the results of each individual we found a constant difference between the increase of alpha 1-antitrypsin and alpha 2-macroglobulin of about 0.030 for the whole range of increases (i.e. for alpha 1-antitrypsin 0-0.15). In contrast, the effects of recumbency were independent of molecular size. The mean, fractional decreases in concentration induced by changing of position from sitting (B) to lying (C) were approximately 0.065 for all three proteins.     

Screening using serum percentage of carbohydrate-deficient transferrin for congenital disorders of glycosylation in children with suspected metabolic disease

BACKGROUND: Diagnoses of congenital disorders of glycosylation (CDG) are based on clinical suspicion and analysis of transferrin (Tf) isoforms. Here we present our experience of CDG screening in children with a suspected metabolic disease by determination of serum percentage of carbohydrate-deficient transferrin (%CDT) in tandem with isoelectric focusing (IEF) analysis of Tf and alpha(1)-antitrypsin (alpha(1)-AT). METHODS: We performed approximately 8000 serum %CDT determinations using %CDT turbidimetric immunoassay (TIA). In selected samples, IEF analysis of Tf and alpha(1)-AT was carried out on an agarose gel (pH 4-8) using an electrophoresis unit. The isoforms were detected by Western blotting and visualized by color development. We performed neuraminidase digestion of serum to detect polymorphic variants of Tf. RESULTS: We established a cutoff value for serum %CDT of 2.5% in our pediatric population. Sixty-five patients showed consistently high values of serum %CDT. In accordance with Tf and alpha(1)-AT IEF profiles, enzyme assays, and mutation analysis, we made the following diagnoses: 23 CDG-Ia, 1 CDG-Ib, and 1 conserved oligomeric Golgi 1 (COG-1) deficiency. In addition, we identified 13 CDG-Ix non Ia, non-Ib; 3 CDG-Ix; and 9 CDG-IIx cases, albeit requiring further characterization; 9 patients with a secondary cause of hypoglycosylation and 6 with a polymorphic Tf variant were also detected. CONCLUSION: The combined use of CDT immunoassay with IEF of Tf and alpha(1)-AT is a useful 1st-line screening tool for identifying CDG patients with an N-glycosylation defect. Additional molecular investigations must of course be carried out to determine the specific genetic disease.     

alpha 2-Macroglobulin in patients with obstructive lung disease, with and without alpha 1-antitrypsin deficiency

Serum alpha 2-macroglobulin concentrations were measured in 178 patients with emphysema and 115 control subjects of similar age and sex distribution. The study group included 59 PI type Z patients with alpha 1-antitrypsin deficiency, five with the rare alpha 1-antitrypsin null genotype (PI Q0 or --), and seven with alpha 1-antitrypsin deficiency of the rare PI types MmaltonZ or MduarteZ. Individuals with all types of alpha 1-antitrypsin deficiency were found to have significantly increased serum concentrations of alpha 2M (p less than 0.001). These increased concentrations were associated with all types of alpha 1-antitrypsin deficiency, not only with the PI type Z. The highest alpha 2-macroglobulin concentrations were found in the PI Q0 patients (5 with emphysema, 2 with no lung disease), and these patients had almost no circulating alpha 1-antitrypsin. Raised concentrations of serum alpha 2-macroglobulin were not due to emphysema: 86 patients with emphysema, of PI type M, and the normal control subjects had similar average concentrations of alpha 2-macroglobulin. One control subject with an average alpha 2-macroglobulin concentration of only 41% of normal was found.