Volume 24, Issue 8, Pages 935-943.e7 (August 2017) Ketone Body Acetoacetate Buffers Methylglyoxal via a Non-enzymatic Conversion during Diabetic and Dietary Ketosis  Trine Salomón, Christian Sibbersen, Jakob Hansen, Dieter Britz, Mads Vandsted Svart, Thomas Schmidt Voss, Niels Møller, Niels Gregersen, Karl Anker Jørgensen, Johan Palmfeldt, Thomas Bjørnskov Poulsen, Mogens Johannsen  Cell Chemical Biology  Volume 24, Issue 8, Pages 935-943.e7 (August 2017) DOI: 10.1016/j.chembiol.2017.07.012 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 935-943. e7DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 In Vivo Reaction between MG and AcAcO Scheme of non-enzymatic in vivo formation of 3-HHD. AcAcO from fatty acid catabolism in the liver reacts with the glycolytic side product MG in the circulatory system, resulting in the formation of 3-HHD. Cell Chemical Biology 2017 24, 935-943.e7DOI: (10.1016/j.chembiol.2017.07.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Comparative Reactivity of AcAcO and Known MG Scavengers In Vitro (A) In vitro kinetic study of the reaction between [2,4-13C2]-AcAcO and MG; here shown for 500 nM MG with 1.5 mM AcAcO. Means of triplicate data of varying initial concentrations of the reactants was used to estimate the rate constant of the reaction (see Figure S1B, Table S3, and Data S1). (B) Extent of [4,6-13C2]-3-HHD formation from MG (1 μM) and [2,4-13C2]-AcAcO in presence of α-aminoguanidine (AG). Means ± SD of triplicates are shown. Further data in Figure S1C. (C) Quantitation of the MG-derived modification of arginine residues, MG-H1, succeeding incubation of human serum albumin (HSA, 10 μM) with MG (500 μM) and AcAcO (1 or 5 mM). Shown are means ± SD of three technical replicates. Further data in Figures S2A–S2C. Cell Chemical Biology 2017 24, 935-943.e7DOI: (10.1016/j.chembiol.2017.07.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Formation and Turnover of 3-HHD in Blood Samples (A) Detection of 3-HHD in non-ketotic controls and ketotic type I diabetic patients. The chromatograms show the MRM transitions of 3-HHD (analyte) along with the isotopically labeled internal standard (IS). (B) 3-HHD formation upon spiking [2,4-13C2]-AcAcO into whole blood (WB)/plasma (PL). Contribution of MG from [2,4-13C2]-AcAcO degradation is distinguished from naturally occurring MG by isotopic labeling of the resulting 3-HHD product (refer to Figure S1A). Means ± SD of three technical replicates are shown. (C) Stability of 3-HHD spiked into whole blood (WB)/plasma (PL). Spiking concentration was 250 nM and means ± SD of three technical replicates are shown (further data in Figures S2E and S2F). Cell Chemical Biology 2017 24, 935-943.e7DOI: (10.1016/j.chembiol.2017.07.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Modification of Human Serum Albumin by 3-HHD Adducts (A) Structures of 3-HHD and HTO (4) and their corresponding lysine adducts. (B) Identification of the HDMP-modified HSA peptide KQTALVELVK (K1: +C6H6O, +94.0419 Da) after reaction of human serum albumin (HSA, 10 μM) with MG (500 μM) and AcAcO (1 or 5 mM). Tryptic peptides were analyzed by nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) and ion chromatograms were extracted (EIC) using the in silico calculated m/z value (611.87) corresponding to the two positively charged modified peptides. (C) An overview of the lysine residues in the HSA amino acid sequence found modified in the study described in Figures S3 and S4. Residues with HDMP modifications are colored red and residues that can be modified by either HDMP and HTO are marked blue. The displayed modifications are those observed in all of three replicate experiments using only highly confident peptide data (mascot p value (expect) < 0.001). (D) Identification of an HTO-derived adduct on an HSA peptide. Tryptic peptides from HSA incubated at 37°C for 24 hr with or without 100 μM HTO were analyzed by nanoLC-MS/MS. The ion chromatogram, corresponding to the peptide KQTALVELVK with the HTO modification at the position 1 lysine residue, was extracted. Cell Chemical Biology 2017 24, 935-943.e7DOI: (10.1016/j.chembiol.2017.07.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 3-HHD Binds and Modify Proteins (A) Scheme illustrating the principle of detection of global 3-HHD protein conjugation and metabolism in whole blood via alk-3-HHD (5) probe and click chemistry. In short, alk-3-HHD (or alkMG) was spiked to a whole-blood sample and incubated for 0–24 hr at 37°C. Following centrifugation, plasma was separated from blood cells and the plasma proteins were precipitated by cold acetone. The protein precipitate as well as proteins from the blood cells were click-conjugated to a fluorophore (rhodamine-azide) and analyzed by SDS-gel and fluorescence scanning (Figures 5B and S5D). Or plasma protein precipitate was click-conjugated to a cleavable azido-azo-biotin tag, digested with trypsin, enriched using streptavidin agarose resin, washed, released, and analyzed by nanoLC-MS/MS (Figure S6 and Table S4). The supernatant-containing soluble alk-3-HHD metabolites were click-conjugated to a solid support (clickable resin), washed, and finally released by chemical cleavage. The metabolites were analyzed by untargeted UPLC-QTOF-MS (Figures 6A and S7A). (B) SDS-PAGE in-gel fluorescence of alk-3-HHD- and alkMG-labeled plasma proteins from whole-blood samples. The Coomassie staining serves as loading control. Varying concentrations of the alkyne-tagged metabolites (0, 1, or 5 mM) were compared. Cell Chemical Biology 2017 24, 935-943.e7DOI: (10.1016/j.chembiol.2017.07.012) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 3-HHD Is Metabolized to Its Di- and Triol in Whole Blood (A) UPLC-QTOF-MS extracted ion chromatograms of alk-3-HHD (blue peak) and its alkyne-tagged metabolite conjugated to the resin residue (green peak). The reduced metabolite appears to dehydrate in the ion source (red and yellow peaks). (B) Time-dependent 3-HHD metabolism in whole blood yielding the singly and doubly reduced metabolites: C6H10O2 and hexane-2,3,5-triol (7), respectively. The major ion traces of the individual metabolites are shown. Cell Chemical Biology 2017 24, 935-943.e7DOI: (10.1016/j.chembiol.2017.07.012) Copyright © 2017 Elsevier Ltd Terms and Conditions