Wernli C, Finochiaro S, Volken C, Andresen-Streichert H, Buettler A, Gygax D, Salomons G S, Jansen E E, Ainslie G R, Vogel K R, Gibson K M
University of Applied Sciences and Arts, Northwestern Switzerland School of Life Sciences, Institute for Chemistry and Bioanalytics, Switzerland.
Bildungszentrum Gesundheit Basel-Stadt, Münchenstein, Switzerland.
Mol Genet Metab Rep. 2016 Aug 17;11:81-89. doi: 10.1016/j.ymgmr.2016.07.009. eCollection 2017 Jun.
An 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.
We 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.
Split 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.
Multiple 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).
We 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.
An 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.
一种用于定量生物流体中γ-羟基丁酸（GHB）的酶法可用于对特定人群进行琥珀酸半醛脱氢酶缺乏症（SSADHD）的靶向筛查。
我们采用了两级研究方法，其中第一项研究（概念验证）检测了7份来自SSADHD患者的尿液样本和5份对照样本，第二项研究（可行性研究）检测了包括血浆在内的更广泛的患者和对照样本群体。
通过酶法、单独的气相色谱法（概念验证）和气相色谱-质谱法对尿液和血浆的分样（匿名）进行评估，并比较结果。
已开发出多种检测GHB的方法。我们评估了一种酶法，该方法采用与NADH产生偶联的重组GHB脱氢酶，后者在Cobas Integra 400 Plus上进行定量。结果：在我们的概念验证研究中，我们分析了12份尿液样本（5份对照，7份SSADHD），在可行性研究中，我们评估了33份尿液样本（23份对照，10份SSADHD）和31份血浆样本（14份对照，17份SSADHD）。在常规临床化学分析仪上进行的酶法检测结果可靠，与尿液中的仪器方法显示出极好的一致性（GC-FID：r = 0.997，p≤0.001；GC-MS：r = 0.99，p≤0.001）；然而，该检测方法略微高估了血浆中的GHB水平，尤其是那些GHB水平较低的样本。相反，对于血浆中较高GHB水平，酶法与比较方法的相关性极好（GC-MS；r = 0.993，p≤0.001）。
我们评估了这种酶法通过定量GHB来识别SSADHD患者的能力。数据表明，酶法可能是一种使用尿液在特定人群中检测SSADHD的合适筛查方法。此外，该检测方法可用于基础研究，以阐明潜在疾病的机制或监测GHB水平以评估候选药物。
对生物流体中GHB的酶法进行评估，作为SSADHD的筛查方法，发现其在尿液中可靠，但应用于血浆时需要改进。
