Determination of ketone bodies in biological samples via rapid UPLC-MS/MS

Puchalska, Patrycja; Nelson, Alisa B.; Stagg, David B.; Crawford, Peter A.

doi:10.1016/j.talanta.2020.122048

Cited by 29 publications

(26 citation statements)

References 55 publications

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“…Among these metabolites, no significant differences were observed between NWT and OWT groups post-exercise. To validate the observations revealed by the semiquantitative untargeted metabolomics pipeline, FFA species and βOHB were formally quantified using validated shotgun lipidomics and targeted UPLC-MS/MS approaches, respectively (52,53) (Supplemental Table 7). These targeted and formally quantitative approaches confirmed those generated through the untargeted metabolomics pipeline: acute aerobic exercise increased βOHB concentrations 2.1-and 3.7-fold in NWT and OWT groups, respectively (exercise effect was statistically significant in both groups, but no significant differences between NWT and OWT groups were observed); and FFA concentrations increased post-exercise between 1.3-4.6-fold in both groups, in which exercise effect was statistically significant in both groups, but with no significant differences between NWT and OWT groups (Supplemental Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Acute aerobic exercise reveals that FAHFAs distinguish the metabolomes of overweight and normal-weight runners

Nelson¹,

Chow²,

Stagg³

et al. 2022

JCI Insight

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Background. Responses of the metabolome to acute aerobic exercise may predict VO2max and longer-term outcomes, including the development of diabetes and its complications.Methods. Serum samples were collected from overweight trained (OWT) and normal weight trained (NWT) runners prior to and immediately after a supervised 90-minute treadmill run at 60% VO2max (NWT = 14, OWT = 11) in a cross-sectional study. We applied a liquid chromatography high resolution-mass spectrometry based untargeted metabolomics platform to evaluate the effect of acute aerobic exercise on the serum metabolome.Results. NWT and OWT metabolic profiles shared increased circulating acylcarnitines and free fatty acids (FFAs) with exercise while intermediates of adenine metabolism, inosine and hypoxanthine, were strongly correlated with body fat percentage and VO2max. Untargeted metabolomics-guided follow-up quantitative lipidomic analysis revealed that baseline levels of fatty acid esters of hydroxy fatty acids (FAHFAs) were generally diminished in the OWT group.FAHFAs negatively correlated with visceral fat mass and HOMA-IR. Strikingly, a 4-fold decrease in FAHFAs was provoked by acute aerobic running in NWT, an effect that negatively correlated with circulating IL-6, neither of which was observed in the OWT group. Machine learning models based on a pre-exercise metabolite profile that included FAHFAs, FFAs, and adenine intermediates predicted VO2max. Conclusion. These findings in overweight human participants and healthy controls indicate that exercise-provoked changes in FAHFAs distinguish normal weight from overweight individuals and could predict VO2max. These results support the notion that FAHFAs could modulate the inflammatory response, fuel utilization, and insulin resistance. Trial registration. ClinicalTrials.gov NCT02150889.

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Section: Resultsmentioning

confidence: 99%

Acute aerobic exercise reveals that FAHFAs distinguish the metabolomes of overweight and normal-weight runners

Nelson¹,

Chow²,

Stagg³

et al. 2022

JCI Insight

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“…AcAc concentration was measured in the cortex and hippocampus collected for 24 h before the assay (Acetoacetate Colorimetric Assay Kit, Sigma Aldrich, MAK199) and stored at –80°C, which allowed the avoidance of its biodegradation ( Puchalska et al, 2021 ). The tissue was homogenized in MilliQ ultrapure water (100 μl per 10 mg of tissue), centrifuged, and used for the assay.…”

Section: Methodsmentioning

confidence: 99%

Differential Response of Hippocampal and Cerebrocortical Autophagy and Ketone Body Metabolism to the Ketogenic Diet

Liśkiewicz

Nowacka-Chmielewska

et al. 2021

Front. Cell. Neurosci.

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Experimental and clinical data support the neuroprotective properties of the ketogenic diet and ketone bodies, but there is still a lot to discover to comprehensively understand the underlying mechanisms. Autophagy is a key mechanism for maintaining cell homeostasis, and therefore its proper function is necessary for preventing accelerated brain aging and neurodegeneration. Due to many potential interconnections, it is possible that the stimulation of autophagy may be one of the mediators of the neuroprotection afforded by the ketogenic diet. Recent studies point to possible interconnections between ketone body metabolism and autophagy. It has been shown that autophagy is essential for hepatic and renal ketogenesis in starvation. On the other hand, exogenous ketone bodies modulate autophagy both in vitro and in vivo. Many regional differences occur between brain structures which concern i.e., metabolic responses and autophagy dynamics. The aim of the present study was to evaluate the influence of the ketogenic diet on autophagic markers and the ketone body utilizing and transporting proteins in the hippocampus and frontal cortex. C57BL/6N male mice were fed with two ketogenic chows composed of fat of either animal or plant origins for 4 weeks. Markers of autophagosome formation as well as proteins associated with ketolysis (BDH1—3-hydroxybutyrate dehydrogenase 1, SCOT/OXCT1—succinyl CoA:3-oxoacid CoA transferase), ketone transport (MCT1—monocarboxylate transporter 1) and ketogenesis (HMGCL, HMGCS2) were measured. The hippocampus showed a robust response to nutritional ketosis in both changes in the markers of autophagy as well as the levels of ketone body utilizing and transporting proteins, which was also accompanied by increased concentrations of ketone bodies in this brain structure, while subtle changes were observed in the frontal cortex. The magnitude of the effects was dependent on the type of ketogenic diet used, suggesting that plant fats may exert a more profound effect on the orchestrated upregulation of autophagy and ketone body metabolism markers. The study provides a foundation for a deeper understanding of the possible interconnections between autophagy and the neuroprotective efficacy of nutritional ketosis.

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“…Tail bleeds were performed before injection and at regular intervals postinjection. Serum ketones were extracted and measured by LC-MS/MS using a recently developed method [ 18 ]. No differences in serum levels of AcAc, βOHB, or total ketone bodies (TKB) were observed before injections (compare the three panels of Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Serum ketones were quantified using a method previously described by our group [ 18 ]. For serum or perfusion effluent (see Section 2.9 ) samples containing unlabeled ketones, 10 μL of diluted serum/perfusion effluent was extracted with 40 μL of cold 1:1 acetonitrile:methanol containing the internal standards [3,4,4,4-D 4 ]βOHB and [U– 13 C 4 ]AcAc, both at 50 μM.…”

Section: Methodsmentioning

confidence: 99%

Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice

Stagg

Gillingham

Nelson

et al. 2021

Molecular Metabolism

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Objective Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. Therefore, we sought to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB). Methods Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, and complementary physiological approaches in novel cell type-specific knockout mice, we quantified the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD + /NADH-dependent oxidation/reduction of ketone bodies. Results Exogenously administered AcAc is reduced to D-βOHB, which increases hepatic NAD + /NADH ratio and reflects hepatic BDH1 activity. Livers of hepatocyte-specific BDH1-deficient mice did not produce D-βOHB, but owing to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte-specific BDH1 deficient mice exhibited diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis, but normal hepatic energy charge overall. Glycemic recovery after acute insulin challenge was impaired in knockout mice, but they were not more susceptible to starvation-induced hypoglycemia. Conclusions Ketone bodies influence liver homeostasis. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production. Therefore, through oxidation/reduction of ketone bodies, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis.

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Determination of ketone bodies in biological samples via rapid UPLC-MS/MS

Cited by 29 publications

References 55 publications

Acute aerobic exercise reveals that FAHFAs distinguish the metabolomes of overweight and normal-weight runners

Acute aerobic exercise reveals that FAHFAs distinguish the metabolomes of overweight and normal-weight runners

Differential Response of Hippocampal and Cerebrocortical Autophagy and Ketone Body Metabolism to the Ketogenic Diet

Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice

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