B-169 Beta-hydroxybutyrate/acetoacetate Ratio as Indicator for Mitochondrial Diseases Utilizing a Novel LC-MS/MS Based Ketone Body Panel

Abstract

Abstract Background Inherited mitochondrial diseases, such as Leigh Syndrome cause defects in the energy metabolism, resulting in abnormalities in the mitochondrial redox state, defined as the ratio between nicotinamide adenine dinucleotide (NADH) and oxidized nicotinamide adenine dinucleotide (NAD+). Accurate and reproducible measurement of the NADH/NAD+ ratio directly is challenging due to its instability. The hepatic intramitochondrial NADH/NAD+ ratio is directly proportional to the ratio between the ketone bodies: beta-hydroxybutyrate (BHB) and acetoacetate (AcAc). Therefore, measuring the ketone body ratio is a more stable approach to estimate the mitochondrial redox state. Due to AcAc stability challenges and the presence of BHB structural isomers (i.e., alpha-hydroxybutyrate (AHB), gamma-hydroxybutyrate (GHB), and beta-hydroxyisobutyrate (BHIB)), no clinically validated assay that measures BHB, AcAc, and their ratio was available as a single test. Existing assays quantify AcAc and BHB separately using spectrophotometry, enzymology, or indirectly via gas chromatography, resulting in significantly reduced accuracy and large specimen quantity requirements. Lack of integration is particularly challenging for calculating a reproducible ratio. To improve clinical care, a novel liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) based ketone body panel requiring only 10 µL of serum or plasma was developed and clinically validated. Methods 10 µL of serum or plasma was taken and kept on ice, followed by a protein precipitation, and drying down of the acquired supernatant. Thereafter, all five analytes were separated using a 6.5 min reversed phased LC column (Acquity, Waters) and detected by MS/MS analysis (Xevo TQ-S, Waters). Data were processed and curated with Ascent V4 (Indigo). Patient samples from the mitochondrial clinic (Children’s Hospital of Philadelphia) were analyzed and results were paired with known diagnoses. Results A new robust and sensitive ketone body panel was developed that not only accurately and reproducibly measures concentrations of BHB, AcAc, and their ratio, but also the BHB isomers AHB, GHB, and BHIB. To ensure quality, a thorough validation and stability study has been performed. All analytes reported a linear range over three orders of magnitude, i.e., 0.0025–1.5 mM (AcAc and AHB), 0.0050–1.5 mM (BHB and BHIB), and 0.0025–1.2 mM (GHB), and a dilution up to 50× is permitted. The precision was assayed over 20× days, 2× replicates, and 2× injections for each analyte at three levels determining the inter-assay (day), intra-assay (replicate), and intra-assay (injection) coefficient of variation (CV); all the %CVs were reported below 6.5%. Spike and recovery were performed to determine the accuracy, results were obtained from ten random patient samples; an average of 99.9% (AcAc), 102.7% (BHB), 95.3% (AHB), 85.7% (GHB), and 87.5% (BHIB) was measured. Additionally, at least 40 samples were cross-examined for BHB using two independent assays, a mean bias of 0.01 mM and a Pearson’s R = 0.996 were reported. Specificity was determined for all analytes against 16 common therapeutic drugs, bilirubin, hemoglobin, triglycerides, and high protein levels at <15% bias. Finally, new reference ranges have been established and were used to calculate accuracy rates. Conclusion Improved mitochondrial disease screening and diagnostics is established utilizing the LC-MS/MS based ketone body panel.