Automated determination of the antitrypsin activity of serum

Hoffmann, J. J. M. L.; Broek, W. G. M. van den; Jansen, A.P.

doi:10.1016/0009-8981(76)90538-6

Cited by 15 publications

(7 citation statements)

References 13 publications

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“…Both proteins wcre determined by a chromogenic subslrate assay (Boehringer Mannheim), the principles of which have bccn described in detail previously (12)(13)(14)(15)18).…”

Section: Funclional Delermination Of U ι-Proteinase Inhibitor Andmentioning

confidence: 99%

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Gressner¹,

Peltzer²

1984

Clinical Chemistry and Laboratory Medicine

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Summary:In sera of healthy persons (n = 50) and patients with a variety of diseases (n = 197) the two major proteinase inhibitors, αι-proteinase inhibitor (oti-antitrypsin) and ai-macroglobulin, were measured by two methods: a chromogenic (amidolytic) Substrate assay to assess the functional activities, and a laser nephelometric method to determine the immunoreactive concentrations of the respective proteins. The specific proteinase inhibitor activities defined s the number of inhibitor units per g inhibitor protein were calculated.The precision and accuracy of both assays were found to be similar, showing a satisfactory correlation of results for the sera of healthy persons (r ?= 0.916 for cta-macroglobulin and 0.988 for cti-proteinase inhibitor). In diseased individuals the correlation was lower than in normal persons (0.862 for a2-macroglobulin and 0.907 for αϊ-proteinase inhibitor). A poor correlation was obtained in patients with liver diseases (r = 0.586 for αι-proteinase inhibitor and 0.852 for ai-macroglobulin).Reference ranges were established for functional and immunological concentrations and for specific inhibitor activities, respectively. Normal values followed a Gaussian distribution.In patients with various diseases including those with acute phase response, the specific inhibitor activities of ctrproteinase inhibitor re reduced significantly; this is because inhibitor activity shows a smaller relative increase than immunoreactivity. Among the various diseases, no significant differences were noted. The specific inhibitor activity of az-macroglobulin changed significantly only in patients with carcinoma, liver diseases and trauma. Follow up of some patients shows also intraindividual Variation of specific proteinase inhibitor activities. Amidolytische und immun-nephelometrische Bestimmung von a/-Proteinase-Inhibitor und

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“…Both proteins wcre determined by a chromogenic subslrate assay (Boehringer Mannheim), the principles of which have bccn described in detail previously (12)(13)(14)(15)18).…”

Section: Funclional Delermination Of U ι-Proteinase Inhibitor Andmentioning

confidence: 99%

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Gressner¹,

Peltzer²

1984

Clinical Chemistry and Laboratory Medicine

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“…Aberrant molecular forms of the molecule have been identified electrophoretically and are called the Pi variants. Hoffman et al 28 described an automated synthetic substrate procedure for α 1 -antitrypsin using the substrate BZ-arg-pNA (BAPNA) and the Vitatron analyzer. The assay was performed in two stages by incubation of serum samples with an excess of trypsin in stage I, and the addition of sub strate and measurement of hydrolyzed pNA in stage II.…”

Section: Antitrypsin Assaysmentioning

confidence: 99%

Development and Clinical Use of Automated Synthetic Substrate Methods for the Evaluation of Coagulation and Fibrinolysis

Mitchell¹

1983

Semin Thromb Hemost

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“…1 Table 9 gives an overview of some existing methods and automated instruments. Besides the enzymes and inhibitors reviewed here, synthetic substrates have also been used for the assay of antitrypsin, 35,56 antielastase, 73 antikallikrein, 2,48 and endotoxin. 83…”

Section: Discussionmentioning

confidence: 99%

Synthetic Peptide Substrate Assays in Coagulation and Fibrinolysis and Their Application on Automates

Friberger¹

1983

Semin Thromb Hemost

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The mammalian proteolytic network involving coagulation, fibrinolytic, kallikrein-kinin, and complement systems is built with inactive zymogens, regulated by activators and inhibitors and by positive as well as negative feedback control mechanisms. They are connected to each other in several ways (Fig. 1). There are a number of components in in blood and vessel tissues that regulate these events.The large number of factors and their numerous points of interaction make their quantitation very difficult. The interpretation of the results obtained is also complicated by the complexity of the systems in question. It was therefore a great step forward when a number of specific synthetic substrates, mainly chromogenic, for protease assays were introduced.A brief description of the biochemistry of the synthetic substrate is included in this article. By utilizing the existing knowledge of the protease systems and considering the different precautions that must be taken, specific methods have been designed. The automation of these methods is then rather a straightforward task. Some principles are discussed here, but each type of equipment has its own characteristic properties. SYNTHETIC PEPTIDE SUBSTRATES Natural Substrates Versus Synthetic SubstratesThe proteases regulating coagulation, fibrinolysis, and other systems (Fig. 1) generally act specifically toward their natural substrates. These substrates are, however, difficult to isolate. The assays in which they are used are often cumbersome and the results are difficult to evaluate. For this reason "the first generation" of synthetic substrates, simple amino acid esters (for example tosoylarginine-methyl ester and benzoyl-arginine-ethyl ester), were introduced. In order to make the assays more simple and sensitive, chromogenic single amide substrates (for example, benzoyl-arginine-pnitroanilide) were developed.A "second generation" of synthetic substrates was born when the single amino acid was exchanged for short peptide chains that primarily were designed to mimic the natural substrate (Fig. 2). Thus it was possible to use affinity sites near the active site of the natural substrate. This was the beginning of more extensive clinical applications of synthetic substrates. The principle of the assay of proteases with a chromogenic tripeptide substrate is shown in Figure 3.The rate at which p-nitroaniline (pNA) is released is measured photometrically at 405 nm, and A A/minute is proportional to the enzyme activity (Michaelis-Menton kinetics). The ultraviolet absorbance curves of intact substrate and of free pNA are shown in Figure 4. The absorbance of the substrate and product at different wavelengths is given in Table 1.Spatial conformation and charge distribution of the substrates are important for the enzyme to recognize its substrate. Some affinity sites of the enzymes may be situated at a distance from the active site (where the reaction takes place) and do not influence the reaction with small molecular weight substrates. This may, on the one hand, be

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Automated determination of the antitrypsin activity of serum

Cited by 15 publications

References 13 publications

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Amidolytic and Immuno-Nephelometric Determination of α1-Proteinase Inhibitor and α2-Macroglobulin in Serum with Calculation of Specific Inhibitor Activities in Health and Disease

Development and Clinical Use of Automated Synthetic Substrate Methods for the Evaluation of Coagulation and Fibrinolysis

Synthetic Peptide Substrate Assays in Coagulation and Fibrinolysis and Their Application on Automates

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