Beta-Galactosidase immunoassay for the measurement of cyclic AMP

The previously described method for phenotyping of alpha₁-antitrypsin (alpha₁-protease inhibitor, Pi) that utilizes separator isoelectric focusing on thin-layer agarose gel (A. R. Qureshi and H. H. Punnett, in Electrophoresis '81, 3rd International Conference on Electrophoresis, pp. 83–87 (1981)) has been improved to give a better resolution of Pi pattern. A shallow pH gradient in the region of the isoelectric point of Pi pattern was obtained by the use of N-(2-acetamido)-2 aminoethanesulfonic acid (1%) and serine (0.8%). The present technique can resolve the Pi alleles. The patterns of Pi phenotypes were found to be similar to those observed on acrylamide gels. The method is fast, reliable, and reproducible.     

Immunologic assay of alpha1-antitrypsin in deficient subjects and their families

Alpha₁-antitrypsin (alpha₁-at) concentration in serum was measured by a method of single radial diffusion. The normal concentration was 212±32 mg/100 ml. Values for members of the families in which the alpha₁-at deficiency occurred were trimodally distributed with values of 25±6, 120±46, and 227±56 mg/100 ml. Subjects with low values were homozygous for the deficiency gene, those with intermediate values were heterozygous, and those with normal values were homozygous for the normal gene.Supported in part by grant HE-06285 from the United States Public Health Service.     

PURIFICATION AND ANALYSIS OF HUMAN ALPHA1-ANTITRYPSIN CONCENTRATE BY A NEW IMMUNOAFFINITY CHROMATOGRAPHY

Alpha₁-antitrypsin is a kind of plasma protein that requires a sequence of different fractionation steps to get generally. To report an effective process for isolating and purifying alpha₁-antitrypsin from Cohn Fraction IV based upon a new immunoaffinity chromatography medium, named “Alpha-1 Antitrypsin Select,” characterization of alpha₁-antitrypsin (α1-AT) was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and tandem mass spectroscopy. Total protein content was determined by the method of Bradford under visible light absorption at 595 nm. Pretreatment process and the immunoaffinity chromatography step achieved a 60.35 ± 1.39% yield. Thus, an overall 71.68 ± 1.32 fold increase in purity and a 41.88 ± 6.98% yield were obtained from plasma. The α1-AT had a specific activity of about 1.00–1.05 PU/mg. This technique will develop an effective process for isolating and purifying, with high purity and specific activity, alpha₁-antitrypsin from Cohn Fraction IV or human whole plasma, which could be an efficient and scaled-up method for alpha₁-antitrypsin products purification.     

Gc,Tf, Pi,PLG, AHS and F13B in two Czech populations

Six serum protein systems, Gc globulin (Gc), transferrin (Tf), alpha₁-antitrypsin (Pi), plasminogen (PLG), alpha2-HS-glycoprotein (AHS), and coaglulation factor XIII B (F13B) have been studied in two Czech populations from Prague (n=243) and Olomouc (n=205). The results are discussed with reference to other investigations in Middle Europe.     

New phenotypes of serum α 1-antitrypsin in Japanese detected by gel slab isoelectric focusing

Summary The phenotype distribution and gene frequencies of serum ₁-antitrypsin in 856 healthy blood donors in Tokyo were examined by gel slab isoelectric focusing (pH 4–6). The allele of the common subtype variant Pi M₂ was present with a frequency of 0.1099 in Japanese. A study of 23 twin pairs and their parents was in agreement with the hypothesis of autosomal codominant inheritance of Pi M subtypes. Other rare variant alleles, Pi M^F, Pi M^S, Pi M^N, Pi M^V, Pi M^X, Pi M^Z were found in very low frequencies.The total concentration of serum ₁-antitrypsin was compared among three different phenotypic groups (M₁, M₁M₂, M₂). Statistically significant quantitative differences were found among these three groups (P<0.01).     

Pharmacokinetic comparability of Prolastin<Superscript>®</Superscript>-C to Prolastin<Superscript>®</Superscript> in alpha<Subscript>1</Subscript>-antitrypsin deficiency: a randomized study

Background

Alpha₁-antitrypsin (AAT) deficiency is characterized by low blood levels of alpha₁-proteinase inhibitor (alpha₁-PI) and may lead to emphysema. Alpha₁-PI protects pulmonary tissue from damage caused by the action of proteolytic enzymes. Augmentation therapy with Prolastin^® (Alpha₁-Proteinase Inhibitor [Human]) to increase the levels of alpha₁-PI has been used to treat individuals with AAT deficiency for over 20 years. Modifications to the Prolastin manufacturing process, incorporating additional purification and pathogen-reduction steps, have led to the development of an alpha₁-PI product, designated Prolastin^®-C (Alpha₁-Proteinase inhibitor [Human]). The pharmacokinetic comparability of Prolastin-C to Prolastin was assessed in subjects with AAT deficiency.

Methods

In total, 24 subjects were randomized to receive 60 mg/kg of functionally active Prolastin-C or Prolastin by weekly intravenous infusion for 8 weeks before crossover to the alternate treatment for another 8 weeks. Pharmacokinetic plasma samples were drawn over 7 days following last dose in the first treatment period and over 10 days following the last dose in the second period. The primary end point for pharmacokinetic comparability was area under the plasma concentration versus time curve over 7 days post dose (AUC_{0-7 days}) of alpha₁-PI determined by potency (functional activity) assay. The crossover phase was followed by an 8-week open-label treatment phase with Prolastin-C only.

Results

Mean AUC_{0-7 days} was 155.9 versus 152.4 mg*h/mL for Prolastin-C and Prolastin, respectively. The geometric least squares mean ratio of AUC_{0-7 days} for Prolastin-C versus Prolastin had a point estimate of 1.03 and a 90% confidence interval of 0.97-1.09, demonstrating pharmacokinetic equivalence between the 2 products. Adverse events were similar for both treatments and occurred at a rate of 0.117 and 0.078 per infusion for Prolastin-C (double-blind treatment phase only) and Prolastin, respectively (p = 0.744). There were no treatment-emergent viral infections in any subject for human immunodeficiency virus, hepatitis B or C, or parvovirus B19 during the course of the study.

Conclusion

Prolastin-C demonstrated pharmacokinetic equivalence and a comparable safety profile to Prolastin.

Trial Registration

ClinicalTrials.gov Identifier: NCT00295061

     

DNA restriction fragment length polymorphisms in differential diagnosis of genetic disease: application in neuromuscular diseases

Summary Three mutations causing alpha-1-antitrypsin defiiency have been identified by gene amplification and direct DNA sequencing. In the Pi (proteinase-inhibitor) null_cardiff gene, the codon for aspartate at position 256 has mutated to encode valine. In Pi M_malton and Pi I, the respective mutations are the deletion of the codon for a phenylalanine residue at position 51 or 52, and a single base substitution resulting in arginine being replaced by cysteine at position 39. Examination of the protein tertiary structure suggests that all of these mutations are likely to result in folding abnormalities that may explain the deficiency states.     

Alpha-1-antitrypsin. Plasma survival studies in the rat of the normal and homozygote deficient forms.

Alpha-1-antitrypsin from normal individuals (Pi type MM) from those with an inherited deficiency of circulatory protein (Pi type ZZ) were labelled with 125I and plasma clearance rates measured in rats either prior to, or following treatment with neuraminidase to remove terminal sialic acid residues. In addition, these proteins and the derivatives were tested for their ability to bind to an hepatic binding protein obtained from rabbit liver membranes that has been shown to be responsible for the clearance of serum asialoglycoproteins. Finally, the two native forms of alpha-1-antitrypsin were treated with galactose oxidase followed by reduction with tritiated potassium borohydride and then analyzed for tritium incorporation in the neutral sugar fraction. The results indicate: (a) clearance from plasma for both forms of alpha-1-antitrypsin is dramatically enhanced upon the loss of terminal sialic acid residues to the liver membrane protein; (b) Z protein does not exhibit terminal galactosyl residues; (c) the low level of Z protein in plasma cannot be accounted for by a faster rate of clearance relative to M protein. The relevance of these findings to the alpha-1-antitrypsin deficiency state are discussed.     

Heterozygosity and localisation of normal allelic fragments for an alpha1-antitrypsin homologous sequence

Summary Approximately 10 kb downstream of the alpha₁-antitrypsin (AAT) gene is a homologous sequence. Two polymorphisms detected with the restriction enzymes Bg1II and EcoRI have been reported previously. We describe two additional polymorphisms with the restriction endonucleases TaqI and HindIII and, for all four restriction enzymes, we have mapped the fragments corresponding to the normal alleles in a cosmid clone.     

Airway wall thickness is increased in COPD patients with bronchodilator responsiveness

Rationale

Bronchodilator responsiveness (BDR) is a common but variable phenomenon in COPD. The CT characteristics of airway dimensions that differentiate COPD subjects with BDR from those without BDR have not been well described. We aimed to assess airway dimensions in COPD subjects with and without BDR.

Methods

We analyzed subjects with GOLD 1–4 disease in the COPDGene® study who had CT airway analysis. We divided patients into two groups: BDR + (post bronchodilator ΔFEV₁ ≥ 10%) and BDR-(post bronchodilator ΔFEV₁ < 10%). The mean wall area percent (WA%) of six segmental bronchi in each subject was quantified using VIDA. Using 3D SLICER, airway wall thickness was also expressed as the square root wall area of an airway of 10 mm (Pi10) and 15 mm (Pi15) diameter. %Emphysema and %gas trapping were also calculated.

Results

2355 subjects in the BDR-group and 1306 in the BDR + group formed our analysis. The BDR + group had a greater Pi10, Pi15, and mean segmental WA% compared to the BDR-group. In multivariate logistic regression using gender, race, current smoking, history of asthma, %emphysema, %gas trapping, %predicted FEV₁, and %predicted FVC, airway wall measures remained independent predictors of BDR. Using a threshold change in FEV₁ ≥ 15% and FEV₁ ≥ 12% and 200 mL to divide patients into groups, the results were similar.

Conclusion

BDR in COPD is independently associated with CT evidence of airway pathology. This study provides us with greater evidence of changes in lung structure that correlate with physiologic manifestations of airflow obstruction in COPD.     

Higher Prevalence of Metformin-Induced Vitamin B12 Deficiency in Sulfonylurea Combination Compared with Insulin Combination in Patients with Type 2 Diabetes: A Cross-Sectional Study

Long-term and high-dose treatment with metformin is known to be associated with vitamin B₁₂ deficiency in patients with type 2 diabetes. We investigated whether the prevalence of B₁₂ deficiency was different in patients treated with different combination of hypoglycemic agents with metformin during the same time period. A total of 394 patients with type 2 diabetes treated with metformin and sulfonylurea (S+M group, n = 299) or metformin and insulin (I+M group, n = 95) were consecutively recruited. The vitamin B₁₂ and folate levels were quantified using the chemiluminescent enzyme immunoassay. Vitamin B₁₂ deficiency was defined as vitamin B₁₂≤300 pg/mL without folate deficiency (folate>4 ng/mL). The mean age of and duration of diabetes in the subjects were 59.4±10.5 years and 12.2±6.7 years, respectively. The mean vitamin B₁₂ level of the total population was 638.0±279.6 pg/mL. The mean serum B₁₂ levels were significantly lower in the S+M group compared with the I+M group (600.0±266.5 vs. 757.7±287.6 pg/mL, P<0.001). The prevalence of vitamin B₁₂ deficiency in the metformin-treated patients was significantly higher in the S+M group compared with the I+M group (17.4% vs. 4.2%, P = 0.001). After adjustment for various factors, such as age, sex, diabetic duration, duration or daily dose of metformin, diabetic complications, and presence of anemia, sulfonylurea use was a significant independent risk factor for B₁₂ deficiency (OR = 4.74, 95% CI 1.41–15.99, P = 0.012). In conclusion, our study demonstrated that patients with type 2 diabetes who were treated with metformin combined with sulfonylurea require clinical attention for vitamin B₁₂ deficiency and regular monitoring of their vitamin B₁₂ levels.     

Pulmonary Emphysema and α1-Antitrypsin Deficiency

Of 72 patients with radiological evidence of pulmonary emphysema, emphysema occurred either alone or in association with bronchitis in 61, and 8 of these (13%) were found to have α₁-antitrypsin deficiency. The main features of this condition are: exertional dyspnoea of relatively early onset (generally between 30 and 45 years of age), severely impaired FEV₁ and T_LCO, and radiological emphysema predominantly affecting the lower zones of the lungs. It is probable that any patient with all the above abnormalities has α₁-antitrypsin deficiency. There is evidence to suggest that cigarette smoking may hasten the onset of this type of emphysema.     

Molecular characterisation of two alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) NullNewport (Gly115→Ser) and (Pi) Z Wrexham (Ser−19→Leu)

Summary Two single point mutations in the alpha-1-antitrypsin gene, resulting in AAT deficiency, have been characterised in heterozygotes by DNA amplification and direct sequencing. The mutations result in amino acid substitutions, Gly¹¹⁵Ser and Ser^–19Leu, in the leader sequence, respectively, and have been designated Pi Null_Newport and Pi Z Wrexham. In the two families studied the mutations occur on chromosomes which also carry the common mutation causing Z deficiency. Individuals with such a deficiency are, therefore, compound heterozygotes. It is not known if these particular mutations would only cause a mild form of AAT deficiency in the absence of the Z mutation as they do not appear to cause predictable folding abnormalities. They do, however, result in severe deficiency when the Z mutation occurs in the same gene.     

Alpha1-antitrypsin levels and electrophoretic patterns of several deficient phenotypes

Summary Two alleles, Pi^Z and Pi^S, are associated with lower than normal alpha₁-antitrypsin concentrations and lower trypsin inhibiting activities in human serum. The phenotypes that result due to heterozygosity or homozygosity for these alleles can be distinguished with electrophoretic procedures. The alpha₁-antitrypsin concentrations of several phenotypes overlap so that the phenotypes can be determined with certainty only by an electrophoretic procedure.

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Zusammenfassung Die zwei Allele Pi^Z and Pi^S führen zu subnormalen Konzentrationen von Alpha₁-Antitrypsin und niedrigen Trypsinhemmaktivitäten im menschlichen Serum. Die durch Hetero- und Homozygotie für diese Allele entstehenden Phänotypen können durch elektrophoretische Methoden unterschieden werden. Die Alpha₁-Antitrypsin-Konzentrationen der verschiedenen Phänotypen überlappen, so daß eine sichere Erkennung nur durch elektrophoretische Methoden möglich ist.

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This work was supported in part by Deutsche Forschungsgemeinschaft and Joachim Jungius-Gesellschaft der Wissenschaften Hamburg.     

Longer Telomere Length in COPD Patients with α1-Antitrypsin Deficiency Independent of Lung Function

Oxidative stress is involved in the pathogenesis of airway obstruction in α₁-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α₁-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α₁-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α₁-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10⁻²⁹). Telomere length was not related to lung function in α₁-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α₁-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.     

Phorbol esters inhibit alpha1 adrenergic stimulation of glycogenolysis in isolated rat hepatocytes

Tumor promoting phorbol esters can stimulate Ca⁺⁺-phospholipid-dependent protein kinase. It has been suggested that this enzyme may mediate the effects of calcium-dependent hormones. In this paper the effects of phorbol 12-myristate 13-acetate (TPA) on isolated rat hepatocyte metabolism were studied. Phorbol esters completely blocked alpha₁-adrenergic stimulation of glycogenolysis. This effect is quite specific for alpha₁-adrenergic actions, as the stimulations of glycogenolysis by vasopressin, angiotensin II, ionophore A-23187 and glucagon were unaffected by TPA. The potencies of the different phorbol esters used in this study suggests that the inhibitory effects of these agents may be due to activation of protein kinase C. The effect of phorbol esters on alpha₁-adrenergic actions seems to occur at an early step of the alpha₁-adrenergic action. TPA (10^?11–10^?6M) was unable to stimulate glycogenolysis. Urea synthesis, which is stimulated by vasopressin and alpha₁-adrenergic agents, was not stimulated by phorbol ester, neither alone nor in combination with the Ca⁺⁺ ionophore A-23187.     

The relationship between phosphate status and photosynthesis in leaves

Spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) were grown in hydroponic culture with varying levels of orthophosphate (Pi). When leaves were fed with 20 mmol·l^–1 Pi at low CO₂ concentrations, a temporary increase of CO₂ uptake was observed in Pi-deficient leaves but not in those from plants grown at 1 mmol·l^–1 Pi. At high concentrations of CO₂ (at 21% or 2% O₂) the Pi-induced stimulation of CO₂ uptake was pronounced in the Pi-deficient leaves. The contents of phosphorylated metabolites in the leaves decreased as a result of Pi deficiency but were restored by Pi feeding. These results demonstrate that there is an appreciable capacity for rapid Pi uptake by leaf mesophyll cells and show that the effects of long-term phosphate deficiency on photosynthesis may be reversed (at least temporarily) within minutes by feeding with Pi.Abbreviation Pi orthophosphate     

Metabolic pathway to 25-hydroxyvitamin D3-26,23-lactone from 25-hydroxyvitamin D3

mRNA was prepared from autopsy liver samples from a homozygote for α₁-antitrypsin deficiency (PiZZ) and from a normal (PiMM) subject. Both preparation gave equivalent synthesis of α₁-antitrypsin in a wheat germ cell-free system. This suggests that the deficiency of plasma α₁-antitrypsin associated with the Z variant is due to a failure of processing and secretion of the protein rather than of its synthesis. It is likely that it is the resultant intracellular accumulation of the Z protein rather than a deficiency of protease inhibitor that is the primary cause of the liver pathology associated with this variant.     

The serum sialyltransferase activity in alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin phenotypes Pi M and Z, purified by the thiol-disulfide exchange procedure, were desialylated by treatment with neuraminidase covalently coupled to Sepharose and used as acceptors of sialic acid in an assay system for serum sialic acid transferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity. Both asialoantitrypsins were equally effective as acceptors in contrast to native Pi Z antitrypsin which did not accept any sialic acid. Serum sialyltransferase activity was determined in 38 adult alpha 1-antitrypsin deficient individuals (Pi Z, MZ, FZ, SZ) with normal liver function and was found to be of the same magnitude as the activity in normal individuals (Pi M). Equal activities were also found in 5 Pi Z patients with cirrhosis of the liver. The results strongly argue against the concept that sialyltransferase deficiency provides the molecular basis for alpha 1-antitrypsin deficiency.     

Leukocyte adhesion and recruitment, and alpha-1-antitrypsin deficiency: a report from ATS 2001, May 18-23, San Francisco

The program at this year's American Thoracic Society international conference included over 300 scientific and clinical symposia. In this report I have reviewed the data presented on two important areas of lung inflammation, namely leukocyte recruitment and alpha-1-antitrypsin deficiency. Highlights included work from a number of groups identifying the contribution of specific leukocyte adhesion molecules (CD18, CD11a and vascular cell adhesion molecule-1) which varied according to the site and nature of the initial inflammatory stimulus. In addition work was presented examining the contribution of various chemoattractants to the process of leukocyte recruitment in chronic obstructive pulmonary disease, with leukotriene B₄ in particular appearing to play a major role. In alpha-1-antitrypsin deficiency other molecules may also be important and work was presented demonstrating the pro-inflammatory potential of alpha-1-antitrypsin polymers in the lungs of these patients. These advances in the understanding of the basic mechanisms of inflammation will, in the future, allow the development of novel anti-inflammatory therapies for a variety of lung diseases.