5-Aminovaleric acid betaine predicts impaired glucose metabolism and diabetes

Background 5-Aminovaleric acid betaine (5-AVAB) has recently been identified as a diet and microbial-dependent factor inducing obesity and hepatic steatosis in mice fed a Western diet. Accumulating evidence suggests a role in metabolic dysfunction associated with obesity, diabetes, and fatty liver disease. However, whether 5-AVAB plays a role in human disease is unclear, and human data are sparse. Methods We measured circulating 5-AVAB serum levels in 143 individuals with overweight or obesity participating in a randomized intervention study (NCT00850629) investigating the long-term effect of a weight maintenance strategy after diet-induced weight reduction. Results Higher 5-AVAB serum levels correlate with worse estimates of obesity, glucose metabolism, and hepatic steatosis after weight loss. Furthermore, higher 5-AVAB levels after weight loss independently predict detrimental changes in glucose metabolism 18 months after the successful weight reduction. Conclusion Our human data supports previous findings in rodents indicating a relevant, potentially disadvantageous function of 5-AVAB in the context of metabolic dysbalance.

The sample preparation was performed according to metaSysX procedure, a modified protocol from 6 Salem et al. [1].Briefly, 50 µl of material was used for extraction of polar and lipophilic compounds by 7 methyl-tert-butyl-ether (MTBE)/methanol/water.The organic phase containing lipids and lipophilic 8 compounds was transferred into the 1.5 ml tube.500 µl and 150 µl of aqueous phase containing semi-9 polar and polar compounds was transferred into the new tubes for LC-and GC-MS analysis, 10 respectively.The collected phases were dried down in vacuum concentrator and stored in -80 °C until 11 analysis.12

LC-MS 13
The dried samples were dissolved in 100 µl of water or acetonitrile for polar and lipid measurements 14 respectively.2 µl were injected for the LC-MS analysis.The analysis was performed using QExactive 15 Orbitrap MS (Thermo Fisher Scientific) coupled to the ultraperformance liquid chromatography 16 (UPLC).17

Liquid phase chromatography 18
The samples were measured with a Waters ACQUITY Reversed Phase Ultra Performance Liquid 19 Chromatography (RP-UPLC) coupled to a Thermo-Fisher QExactive mass spectrometer.BEH 20 C8 (ACQUITY UPLC BEH Column, 130Å, 1.7 µm, 2.1 mm X 100 mm) and HSS T3 C18 (ACQUITY 21 UPLC HSS T3 Column, 100Å, 1.8 µm, 2.1 mm X 100 mm) columns were used for the lipophilic and 22 the hydrophilic measurements, respectively.A 15 min gradient was used for separation of polar and 23 lipophilic compounds.The mobile phases for separation of polar and semi-polar compounds were 0.1% were used for chromatographic separation: A 95% to A 60% to 11 min, A 60% to A 30% from 11 to 13 26 min and A30% to A 1% from 13 to 15 min.For the separation of lipids and lipophilic metabolites 27 following mobile phases were used: 1% of 1M NH4Ac in 0.1% acetic acid (buffer A) and acetonitrile: 28 isopropanol (7:3) containing 1% of 1M NH4Ac in 0.1% acetic acid (buffer B) with the step gradient of 29 45% to 25 % A from 1 to 4 min, 25% to 11% A from 4 to 12 min and 11% to 0% A from 12 to 15 min.30

Mass spectrometry 31
All mass spectra were acquired in Full Scan MS (Mass Range [100-1500]) positive and negative 32 ionization mode with the following settings of the instrument: Heated electrospray ionization (HESI) 33 was used, spray voltage was 3.5 kV, capillary temperature 275 °C, sheath gas flow rate 60 units, mass 34 resolving power 70000, 3e6 target value (AGC) and maximal fill time of 200 ms.. To increase number 35 of identified compounds pooled samples were measured in LC-MS/MS mode.The full mass spectrum 36 was acquired for mass range 100-1500 every three MS/MS scans that were recorder for mass range 100-37 1500.The higher-energy collisional dissociation (HCD) fragmentation spectrum was recorded for three 38 most intense precursor ions at normalized collision energy 25.The mass resolution was set to 35000 and 39 17500, the AGC to 1e5 and 5e4 ions, and maximal fill time of 100 ms and 50 ms for MS and MS/MS, 40 respectively.The dynamic exclusion was set to 3s.Thermo Excalibur was used for the data acquisition.41

LC-MS data annotation 42
metaSysX database of chemical compounds was used to annotate the features detected in the LC-MS 43 polar and non-polar platform.The metaSysX database contains the mass-to-charge ratio and the 44 retention time information of reference compounds measured at the same chromatographic and 45 spectrometric condition as samples measurements.7 ppm and 0.1 min deviation from the reference 46 compounds mass-to-charge-ratio and retention time were allowed as matching criteria for polar and lipid 47 platform.Coeluting compounds with the same mass of generated ion were kept as conflicting annotation.48 Annotated lipids were confirmed using R-based algorithm developed in metaSysX which uses MS/MS 49 fragmentation spectrum.50 51 Reference list