Lactobionate

At the two measurement points, in addition to recording of blood temperature, heart rate and MAP, arterial blood samples were taken for the measurement of PaO₂ and PaCO₂, acid-base-status, electrolyte (Na⁺, K⁺) levels and metabolic parameters (lactate, glucose). These parameters were measured using the automatic blood gas analyzer which not only provides blood gas tensions but also acid-base status, electrolyte concentrations as well as lactatemia and glycemia. Furthermore, at the first blood sampling after the 30-min recovery period, a blood cell count was obtained with the help of a Neubauer chamber and an Automated Cell Counter (BioRad TC20) corrected for swine species-specific cell size. At the same time-point, cytokines (tumor necrosis factor, interleukin-6 and 10), renin, aldosterone, troponin, testosterone, ACTH and “brain injury markers” were measured using pig-specific commercially available kits (cytokines: Quantikine ELISA Porcine, R&D Systems; Troponin: Pig Cardiac Troponin I, Life Diagnostics; Testosterone: Pig Testosterone, Abnova; ACTH: Pig ACTH ELISA kit, Abbexa; brain markers, Renin, Aldosterone: Porcine ELISA kits, BlueGene BioTech). 8-Isoprotane was determined with an EIA kit (Cayman Chemical). Cortisol values were determined with a commercially available kit for human plasma (IBL International), since it does not differ between species. Catecholamine levels (adrenaline, noradrenaline) were determined after centrifugation of whole blood samples in Li⁺-heparine-coated, stabilizor-primed (20 μL/mL blood containing 0.2 M reduced glutathione and 0.2 M ethylenglycol-bis(aminoethylether)-N,N,N′,N′-tetra-acetic acid (EGTA), both Carl-Roth, Karlsruhe) tubes using liquid-chromatography/tandem-mass-spectrometry (LC-MS/MS) by a service lab (Dr. Eberhard & Partner, Dortmund).
Isolation of peripheral blood mononuclear cells (PBMC) and granulocytes was performed as described recently (Wolfschmitt et al., 2023 (link)). For this purpose, approx. 300 mL of arterial whole blood were collected in 10 mL-LiHep monovettes (Sarstedt, Nümbrecht) from the catheter placed in the left iliac artery. After sampling, the blood was diluted 1:1 with phosphate-buffered saline (PBS; without CaCl₂, MgCl₂) and carefully layered onto two density gradient solutions (9 mL 1.119 g/L and 8 mL 1.088 g/L solution, Pancoll, PAN Biotech, Aidenbach) before centrifugation at 764 g for 20 min without break at room temperature (RT). Density centrifugation yielded a buffy coat containing PBMCs and a bottom layer comprising red blood cells (RBCs) and granulocytes. After washing with 1× PBS, the buffy coat was subjected to osmotic lysis to remove residual RBCs. The lysis was stopped with 10× PBS, and PBMCs were washed and resuspended in 1× PBS before counting cells in a Neubauer counting chamber. In order to purify granulocytes from the bottom layer of the density gradient solution, osmotic lysis had to be performed a total of three times to eliminate RBC contamination. Analogously to PBMCs, granulocytes were washed with 1× PBS after lysis and subsequently counted.
Mitochondrial respiration in the isolated immune cells as well as the brain tissue specimens (pre-frontal cortex) was measured by high-resolution respirometry using the Oxygraph-2K^® (Oroboros Instruments, Innsbruck) as described previously (Datzmann et al., 2023 (link); Datzmann et al., 2021 (link); Zink et al., 2021 (link); Wolfschmitt et al., 2023 (link)) This device allows for simultaneous recording of the O₂ concentration in two parallel chambers calibrated for 2 mL of respiration medium MiR05 (Doerrier et al., 2018 (link); Zink et al., 2021 (link)). This medium is composed of 110 mM D-Sucrose (Sigma Aldrich, St. Louis, MO, United States), 60 mM K-Lactobionate (Sigma Aldrich, St. Louis, MO), 0.5 mM ethylene glycol tetra acetic acid (Sigma Aldrich, St. Louis, MO), 1 g/L bovine serum albumin free from essentially fatty acids (Sigma Aldrich, St. Louis, MO), 3 mM MgCl₂ (Scharlau, Hamburg), 20 mM taurine (Sigma Aldrich, St. Louis, MO), 10 mM KH₂PO₄ (Merck, Darmstadt), 20 mM HEPES (Sigma Aldrich, St. Louis, MO), adjusted to pH = 7.1 with KOH and equilibrated with 21% O₂ at 37°C. Cell suspensions or brain tissue homogenates containing 10 × 10⁶ cells/mL or 1 mg tissue/mL of respiration medium were filled into both chambers and continuously stirred at 750 rpm. Closing the chambers by gently pushing down the stoppers started the continuous recording of mitochondrial respiration, which was quantified in terms of O₂ flux (JO₂) based on the rate of change of the O₂-concentration in the chambers normalized for cell number or tissue weight. Once the chambers were sealed, specific analysis of mitochondrial respiratory function was achieved by sequential injections of substrates and inhibitors into the respiration medium. For blood cell samples, firstly, routine respiration was recorded once a stable JO₂-value was achieved after closing the chambers. Afterwards, 2.5 μM oligomycin was injected to block the ATP-synthase. This yielded the LEAK-state, which represents the respiratory activity required to maintain a stable membrane potential in absence of ATP-turnover. Thereafter, the titration of carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP) in 1 µM steps allowed to achieve the respiratory activity in the un-coupled state. The maximum respiratory capacity (ETC.) was determined after the addition of 2 mM malate, 10 mM glutamate, 5 mM ADP, 5 µM cytochrome c, 10 mM pyruvate and 10 mM succinate. Finally, 0.5 μM rotenone +5 μM antimycin were added to block complex I and III respectively, yielding the residual (non-mitochondrial) O₂ consumption. For tissue samples, the maximum respiratory capacity in the coupled state (OxPhos) was determined after the addition of 2 mM malate, 10 mM glutamate, 5 mM ADP, 5 µM cytochrome c, 10 mM pyruvate, 1 mM octanoyl-carnitine and 10 mM succinate. The LEAK state was recorded after the addition of 2.5 µM oligomycine, and the maximum respiratory capacity in the uncoupled state (ETC.) was measured after the titration of 1 µM FCCP. The data shown are normalized for tissue wet weight and cell number for the brain specimens and isolated blood immune cell count, respectively. Finally, the “Bioenergetic Health Index (BHI)" was calculated as described by Chacko et al. (2014) (link) according to the formula: $BHI=\log \frac{\left(reservecapacity\times ATP-linked{O}_{2}consumption\right)}{\left(non-mitochondrial{O}_{2}consumption\times LEAKrespiration\right)}$
Whole blood superoxide anion (O₂^•−) concentrations were determined immediately after sampling as described previously (Zink et al., 2021 (link); Datzmann et al., 2022 (link); Datzmann et al., 2023 (link)). For this purpose, 25 µL of whole blood were mixed with an aliquot of 25 µL freshly thawed CMH spin probe solution. The CMH solution contained 400 µM CMH spin probe (1-Hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine), 25 µM deferoxamine, and 5 µM diethyldithiocarbamate to chelate transition metal ions in Krebs-HEPES-Buffer (KHB) (Noxygen, Elzach, Germany). After mixing whole blood with CMH, the solution was transferred to a 50 µL glass capillary, sealed, and measured with an EMXnano electron spin resonance (ESR) spectrometer (Bruker, Billerica, MA, United States) after 5 min incubation at 37°C (Bio-III, Noxygen, Elzach, Germany). For each measurement, 3 scans of the following settings were averaged: 3440 G center field, 60 G sweep width, 72.70 ms conversion time, 9.66 GHz microwave frequency, 0.3162 mW microwave power, and 2 G modulation amplitude. Radical concentration was quantified by comparison with a series of CP^• radical standards solved in KHB. As a blank sample, KHB added to the respective amount of CMH was measured and subtracted from the sample value. For the determination of immune cell O₂^•− production (Wolfschmitt et al., 2023 (link)), 25 µL of a cell suspension containing 2.5 × 106 cells/mL purified granulocytes and mononuclear cells were transferred to FACS tubes and washed in RPMI 1640 medium (Glucose 1.8 mg/mL, Glutamine 0.6 mg/mL, NaHCO₃ 100 μg/mL, HEPES 20 mM) while being kept on ice. 50 μL of 1 mg/mL pHrodo^®E. coli BioParticles^® Conjugate (Life technologies TM Carlsbad, CA) was added to the cell suspension before subsequent incubation at 37°C for exactly 20 min. Reactions were stopped by transferring cells on ice and adding 1 mL of washing buffer (pHrodoTM Bioparticles Phagocytosis Kit for Flow Cytometry, Life technologies TM Carlsbad, CA). PBMCs and granulocytes were washed once with PBS before being transferred to a 1.5 mL tube and being resuspended in 1 mL RPMI. For the subsequent ESR analysis, the cell suspension was mixed with 25 µL of CMH. In contrast to whole blood, cell samples were measured 8 times over 30 min to calculate the O₂^•− production rate. Otherwise, the ESR settings were the same as used for a single whole blood measurement. A sample of RPMI 1640 medium mixed 1:1 with CMH was used as a blank value for measuring cell suspensions and subtracted from sample values. Data were evaluated with the Xenon_nano software (Bruker BioSpin, Rheinstetten).

Rat cardiomyocytes were isolated as in Sepp et al. (2010) (link). Live cells were imaged using the approach similar to Birkedal et al. (2006) (link) and Vendelin & Birkedal (2008) (link). In short, the cells were kept and imaged in solution consisting of (mM): KH₂PO₄ 3, MgCl₂ 3, sucrose 110, K-lactobionate 60, taurine 20, HEPES 20, EGTA 0.5, DTT 0.5, malate 2, glutamate 5 and 5 mg mL⁻¹ BSA. pH was adjusted to 7.1 with KOH at 25°. Mitochondria were visualized by staining isolated cells with MitoTracker Green FM with the final concentration of 200 nm; for sarcolemma we used di-8-ANEPPS with the final concentration of 1 μM (both from Invitrogen). After incubation for 15 min at the room temperature, cells were washed and inserted to imaging chamber that consisted of a FlexiPERM silicone insert (Vivascience, Hanau, Germany) attached to a cover slip glass of 0.17 mm in thickness.
For acquisition of images, the same microscope set-ups were used as described earlier for measuring PSF.

We studied the mitochondrial respiration, specifically, oxygen consumption, of ex vivo tissue samples and two colon cancer cell lines harboring BRAF mutations (HT-29 and WiDr) using the high-resolution respirometry system Oxygraph-2k (OROBOROS Instruments, Innsbruck, Austria) at 37 °C. The medium for the measurements contained 0.5 mM of EGTA, 3 mM of MgCl₂, 60 mM of K-lactobionate, 20 mM of taurine, 10 mM of KH₂PO₄, 20 mM of HEPES, and 110 mM of sucrose, adjusted to pH 7.1 at 37 °C. To permeabilize the cell membranes, 16 μg/mL of digitonin was added. We documented the non-phosphorylating state 2 (V0) respiration rate in the medium containing the tissue samples, mitochondrial complex I substrates (5 mM of glutamate + 2 mM of malate), and the complex II substrate succinate (12 mM). After adding 1 mM of ADP, we assessed the state 3 respiration rate (VADP). All respiration rates were normalized to the dry weight of the tissue in milligrams.