An Enzymatic Method to Determine γ-Hydroxybutyric Acid in Serum and Urine

Hasan, Lara; Jermann, Thomas M.; Weber, Jakob; Abrahamsson, L; Sciotti, Michel-Angelo; Böttcher, Michael; Jöchle, W.; Gygax, Daniel; Scholer, André

doi:10.1097/ftd.0b013e318239a41a

Cited by 19 publications

(15 citation statements)

References 38 publications

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“…GHB was quantified on a Cobas Integra 400 Plus [14]. Calibrators (lyophilized GHB in water, Lot 3815 10 mg/L and 100 mg/L) and controls (lyophilized GHB in human urine, Lot 3815: low control 12.6 mg/L and high control 68 mg/L GHB) were provided by Buhlmann laboratories AG and reconstituted with 2 mL deionized water for use.…”

Section: Methodsmentioning

confidence: 99%

“…The standard curve for NADH production was measured with two calibrators described above in duplicate, and was valid for 14 days. Although analytical sensitivity, imprecision, recoveries, and assay linearity have been previously reported [14], these parameters were re-evaluated in the current study.…”

Section: Methodsmentioning

confidence: 99%

“…The lower limit of quantification (LLOQ) for serum is stated as 4.5 mg/l (~ 43.2 μmol/L) and for urine 2.8 mg/l (29.6 μmol/L) [14]. For qualitative evaluation in a screening setting the LOD can be used to decide if the sample is positive or negative for elevated GHB- levels.…”

Section: Methodsmentioning

confidence: 99%

“…intoxication), and it can be run on clinical chemistry analyzers which are generally available around the clock. This assay employs recombinant GHB dehydrogenase (GHBDH, EC 1.1.1.61), which catalyzes the oxidation of GHB to succinic semialdehyde with stoichiometric production of NADH which is quantified spectrophotometrically at 340 nm [13], [14]. Here, we have evaluated the capacity of this enzymatic assay to identify patients with SSADHD via quantitation of GHB.…”

Section: Introductionmentioning

confidence: 99%

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Targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) employing an enzymatic assay for γ-hydroxybutyric acid (GHB) in biofluids

Wernli

Finochiaro

Volken

et al. 2017

Molecular Genetics and Metabolism Reports

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HypothesisAn enzymatic assay for quantification of γ-hydroxybutyric acid (GHB) in biofluids can be employed for targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) in selected populations.RationaleWe used a two-tiered study approach, in which the first study (proof of concept) examined 7 urine samples derived from patients with SSADHD and 5 controls, and the second study (feasibility study) examined a broader sample population of patients and controls, including plasma.ObjectiveSplit samples of urine and plasma (anonymized) were evaluated by enzymatic assay, gas chromatography alone (proof of concept) and gas chromatography–mass spectrometry, and the results compared.MethodMultiple detection methods have been developed to detect GHB. We evaluated an enzymatic assay which employs recombinant GHB dehydrogenase coupled to NADH production, the latter quantified on a Cobas Integra 400 Plus. Results: In our proof of concept study, we analyzed 12 urine samples (5 controls, 7 SSADHD), and in the feasibility study we evaluated 33 urine samples (23 controls, 10 SSADHD) and 31 plasma samples (14 controls, 17 SSADHD). The enzymatic assay carried out on a routine clinical chemistry analyzer was robust, revealing excellent agreement with instrumental methods in urine (GC-FID: r = 0.997, p ≤ 0.001; GC–MS: r = 0.99, p ≤ 0.001); however, the assay slightly over-estimated GHB levels in plasma, especially those in which GHB levels were low. Conversely, correlations for the enzymatic assay with comparator methods for higher plasma GHB levels were excellent (GC–MS; r = 0.993, p ≤ 0.001).ConclusionWe have evaluated the capacity of this enzymatic assay to identify patients with SSADHD via quantitation of GHB. The data suggests that the enzymatic assay may be a suitable screening method to detect SSADHD in selected populations using urine. In addition, the assay can be used in basic research the elucidate the mechanism of the underlying disease or monitor GHB- levels for the evaluation of drug candidates.SynopsisAn enzymatic assay for GHB in biofluids was evaluated as a screening method for SSADHD and found to be reliable in urine, but in need of refinement for application to plasma.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) employing an enzymatic assay for γ-hydroxybutyric acid (GHB) in biofluids

Wernli

Finochiaro

Volken

et al. 2017

Molecular Genetics and Metabolism Reports

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“…We therefore implemented the only available enzymatic assay by Bühlmann laboratories AG. 3 Within 30 minutes, this rapid screening determines the concentration of GHB in the sample. For this assay, a cut-off value is applied to discriminate between the endogenous and exogenous levels of GHB.…”

Section: Therapeutic Drug Monitoring Consultantmentioning

confidence: 99%

Intoxication of a Young Girl Reveals the Pitfalls of GHB Rapid Screening

Franken

Andrews

Slooff

et al. 2016

Therapeutic Drug Monitoring

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The authors discuss the case of a 14-year-old girl who was transferred to the ICU of our hospital with ethanol intoxication (3.3 g/L), loss of consciousness (E5M3V1), and severe amnesia on recovery that was suspected of gamma-hydroxybutyric acid (GHB) intoxication. STAT toxicology screening may be necessary, when sexual assault under GHB intoxication is suspected. Therefore, the initial analysis of a urine sample was performed with a new enzymatic assay analysis for GHB. The enzymatic assay reported a GHB concentration of 26 mg/L, which is above the cut-off value of 10 mg/L. This cut-off value is to differentiate endogenous and exogenous levels because low levels of GHB occur naturally in the body. However, confirmation of these results by gas chromatography, which is common practice to confirm a positive GHB, gave a negative result. This discrepancy is probably contributed to interference of ethanol with the assay. This is a substantial downside of the GHB rapid screening, since the combination of GHB and ethanol is common. It is therefore advised to confirm that the positive GHB results are lower than 50 mg/L by gas chromatography, when using the rapid screening. This way the false-positive results and consequent inappropriate social and legal actions may be avoided.

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Screening and confirmation methods for GHB determination in biological fluids

et al. 2014

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The purpose of this review is to provide a comprehensive overview of reported methods for screening and confirmation of the low-molecular-weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polarity of the compound, its endogenous presence, its rapid metabolism after ingestion, and its instability during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone) are challenges for the analyst and for interpretation of a positive result. First, possible screening procedures for GHB are discussed, including colorimetric, enzymatic, and chromatography-based procedures. Confirmation methods for clinical and forensic cases mostly involve gas chromatography (coupled to mass spectrometry), although liquid chromatography and capillary zone electrophoresis have also been used. Before injection, sample-preparation techniques include (a combination of) liquid-liquid, solid-phase, or headspace extraction, and chemical modification of the polar compound. Also simple "dilute-and-shoot" may be sufficient for urine or serum. Advantages, limitations, and trends are discussed.

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An Enzymatic Method to Determine γ-Hydroxybutyric Acid in Serum and Urine

Abstract: This automated GHB assay is fully quantitative and allows the accurate measurement of GHB in serum and urine. It can be used as a rapid screening assay for the determination of GHB in intoxicated or overdosed patients.

Cited by 19 publications

References 38 publications

Targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) employing an enzymatic assay for γ-hydroxybutyric acid (GHB) in biofluids

Targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) employing an enzymatic assay for γ-hydroxybutyric acid (GHB) in biofluids

Intoxication of a Young Girl Reveals the Pitfalls of GHB Rapid Screening

Screening and confirmation methods for GHB determination in biological fluids

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