Topspin 2

Lipids were extracted from 0.5 ml serum by Folch Solvent (1 ml each of CDCl3 MeOD and CsEDTA (0.2 M), pH 8). After centrifugation, the lower layer was analyzed at 600 MHz cQNP using a NMR spectrometer Avance III 600 (Bruker, Karlsruhe, D), magnetic flux density 14.1 Tesla, a QNP cryo probe, and automated sample changer Bruker B-ACS 120. Computer Intel Core2 Duo 2.4 GHz under MS Windows XP and Bruker TopSpin 2.1 was used for acquisition, while Bruker TopSpin 2.1 was used for processing [47 -50 ].

Initial culture media and culture samples collected at different times were filtered (Minisart 0.2 µM filter from Sartorius, Göttingen, Germany). Supernatant fraction were prepared for 1D 1H NMR analysis, by adding 100 μL of Deuterated trimethylsilyl propionate (TSPd4) at 4,3 mM diluted in DH₂O to 500 μL of supernatant. 1D 1H NMR spectra were acquired on a Bruker Ascend 800 MHz magnet (Bruker, Germany) using a 5 mm CPQCI cryoprobe 1H-31/13C/15 N/Z GRD. A sequence using presaturation (ZGPR) was used for water signal suppression, with a 30° pulse angle and a relaxation delay between scans of 10 s to ensure full signal recovery. A total of 32 scans were accumulated (after 4 dummy scans) with 292 K data points, 6.83 s of acquisition time, 5 s of recycle delay and no spin. Using Topspin 2.1 (Bruker, Rheinstatten, Germany), the FIDs were zero-filled, Fourier transformed with 0.5-Hz exponential line broadening, manually phase corrected, automatically baseline corrected, and aligned to the TSPd4 signal. Topspin 2.1 (Bruker, Rheinstatten, Germany) was also used for peak integration. Metabolite quantification was performed using a program developed in R [30 ]. Three samples were collected and analyzed for each dilution rate.

A sample of purified K82 CPS was deuterium exchanged and examined as a solution in 99.95% D₂O on a Bruker Avance II 600 MHz spectrometer (Bruker Daltonics, Bremen, Germany). Sodium 3-trimethylsilylpropanoate-2,2,3,3-d₄ (δ_H 0, δ_C −1.6) was used as an internal reference for calibration. Two-dimensional ¹H-¹H correlation spectroscopy (COSY), ¹H-¹H total correlation spectroscopy (TOCSY), ¹H-¹H rotating-frame nuclear Overhauser effect spectroscopy (ROESY), ¹H-¹³C heteronuclear single-quantum coherence (HSQC), and ¹H-¹³C heteronuclear multiple-bond correlation (HMBC) experiments were performed using standard Bruker software (Bruker TopSpin 3.6.0 program). The Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A spin lock time of 60 ms and mixing time of 200 ms were used in ¹H-¹H TOCSY and ¹H-¹H ROESY experiments, respectively. A ¹H-¹³C HMBC experiment was recorded, with a 60 ms delay for the evolution of long-range couplings, to optimize the spectrum for coupling constant J_H,C 8 Hz.