A modified, optimized kinetic photometric assay for the determination of blood coagulation factor XIII activity in plasma

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Abstract

Background: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca2+. FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. Methods: In the assay, FXIII was activated by thrombin and Ca2+. Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogehase and NADPH. Results: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was

Original languageEnglish
Pages (from-to)1946-1955
Number of pages10
JournalClinical Chemistry
Volume46
Issue number12
Publication statusPublished - 2000

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Factor XIII
Assays
Plasmas
Kinetics
Fibrin
Transglutaminases
Glutamine
NADP
Thrombin
Factor XIIIa
Substrates
Enzyme Precursors
Ammonia
Polymerization
NAD
Limit of Detection
Glutamic Acid
Substitution reactions
Stabilization
Recovery

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

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title = "A modified, optimized kinetic photometric assay for the determination of blood coagulation factor XIII activity in plasma",
abstract = "Background: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca2+. FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. Methods: In the assay, FXIII was activated by thrombin and Ca2+. Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogehase and NADPH. Results: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96{\%}, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was",
author = "L. K{\'a}rp{\'a}ti and B. Penke and E. Katona and I. Balogh and G. V{\'a}mosi and L. Muszbek",
year = "2000",
language = "English",
volume = "46",
pages = "1946--1955",
journal = "Clinical Chemistry",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "12",

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TY - JOUR

T1 - A modified, optimized kinetic photometric assay for the determination of blood coagulation factor XIII activity in plasma

AU - Kárpáti, L.

AU - Penke, B.

AU - Katona, E.

AU - Balogh, I.

AU - Vámosi, G.

AU - Muszbek, L.

PY - 2000

Y1 - 2000

N2 - Background: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca2+. FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. Methods: In the assay, FXIII was activated by thrombin and Ca2+. Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogehase and NADPH. Results: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was

AB - Background: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca2+. FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. Methods: In the assay, FXIII was activated by thrombin and Ca2+. Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogehase and NADPH. Results: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was

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JO - Clinical Chemistry

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