Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid
Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | deu |
| Published: |
Swiss Chemical Society
2010-11-01
|
| Series: | CHIMIA |
| Subjects: | |
| Online Access: | https://www.chimia.ch/chimia/article/view/4924 |
| _version_ | 1819278805456388096 |
|---|---|
| author | Michel A. Sciotti Lara Hasan André Scholer Thomas M. Jermann Jakob M. Weber Daniel Gygaxa |
| author_facet | Michel A. Sciotti Lara Hasan André Scholer Thomas M. Jermann Jakob M. Weber Daniel Gygaxa |
| author_sort | Michel A. Sciotti |
| collection | DOAJ |
| description |
Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for
the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein
in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in
physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+.
With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a KM of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries
of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.
|
| first_indexed | 2024-12-24T00:17:51Z |
| format | Article |
| id | doaj.art-405ccbf1da6845849ad4084474946251 |
| institution | Directory Open Access Journal |
| issn | 0009-4293 2673-2424 |
| language | deu |
| last_indexed | 2024-12-24T00:17:51Z |
| publishDate | 2010-11-01 |
| publisher | Swiss Chemical Society |
| record_format | Article |
| series | CHIMIA |
| spelling | doaj.art-405ccbf1da6845849ad40844749462512022-12-21T17:24:41ZdeuSwiss Chemical SocietyCHIMIA0009-42932673-24242010-11-01641110.2533/chimia.2010.793Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric AcidMichel A. Sciotti0Lara Hasan1André Scholer2Thomas M. JermannJakob M. Weber3Daniel Gygaxa4Hochschule für Life Sciences Fachhochschule Nordwestschweiz Gründenstrasse 40 CH-4132 Muttenz;, Email: michelangelo.sciotti@fhnw.chBühlmann Laboratories AG Baselstrasse 55 CH-4124 SchönenbuchLabormedizin Universitätsspital Basel CH-4031 BaselBühlmann Laboratories AG Baselstrasse 55 CH-4124 SchönenbuchHochschule für Life Sciences Fachhochschule Nordwestschweiz Gründenstrasse 40 CH-4132 Muttenz; Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a KM of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively. https://www.chimia.ch/chimia/article/view/4924Enzymatic assayGamma hydroxybutyric acidGhbGhb dehydrogenaseReaction mechanism |
| spellingShingle | Michel A. Sciotti Lara Hasan André Scholer Thomas M. Jermann Jakob M. Weber Daniel Gygaxa Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid CHIMIA Enzymatic assay Gamma hydroxybutyric acid Ghb Ghb dehydrogenase Reaction mechanism |
| title | Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid |
| title_full | Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid |
| title_fullStr | Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid |
| title_full_unstemmed | Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid |
| title_short | Development and Characterization of an Enzymatic Method for the Rapid Determination of Gamma Hydroxybutyric Acid |
| title_sort | development and characterization of an enzymatic method for the rapid determination of gamma hydroxybutyric acid |
| topic | Enzymatic assay Gamma hydroxybutyric acid Ghb Ghb dehydrogenase Reaction mechanism |
| url | https://www.chimia.ch/chimia/article/view/4924 |
| work_keys_str_mv | AT michelasciotti developmentandcharacterizationofanenzymaticmethodfortherapiddeterminationofgammahydroxybutyricacid AT larahasan developmentandcharacterizationofanenzymaticmethodfortherapiddeterminationofgammahydroxybutyricacid AT andrescholer developmentandcharacterizationofanenzymaticmethodfortherapiddeterminationofgammahydroxybutyricacid AT thomasmjermann developmentandcharacterizationofanenzymaticmethodfortherapiddeterminationofgammahydroxybutyricacid AT jakobmweber developmentandcharacterizationofanenzymaticmethodfortherapiddeterminationofgammahydroxybutyricacid AT danielgygaxa developmentandcharacterizationofanenzymaticmethodfortherapiddeterminationofgammahydroxybutyricacid |