Supplementary 1
LC/MS/MS Method
Five NEFAs in plasma, including palmitic, stearic, oleic, palmitoleic and linoleic acid, were measured using liquid chromatography-mass spectrometry (LC-MS/MS). One hundred microliters of blood sample was removed from the -80°C freezer and defrosted completely at room temperature. Margaric acid (as an internal standard) was added to the blood to give a final concentration of 20 ppm. Dole’s extraction was performed to separate NEFAs from the plasma at over 95% recovery. The procedure was as follows: 100 uL of serum was mixed with 20 µL of heptadecanoic (margaric) acid in isopropanol (5 µmol/L) as internal standard (included also in the added fatty acid mixture in recovery experiments) in a 1.5-mL Reacti-vial. 500 µL of the modified Dole’s mixture: isopropanol, n-heptane/phosphoric acid (2 mol/ L), 40/10/1 (by vol) was added and mixed again. After 5-10 min of incubation at room temperature, 200 µL of n-heptane and 300 uL of water were added, thoroughly vortex-mixed, and centrifuged for 5 min at 1,000 g. 200 µL of the upper organic layer (i.e., 62.5% of the total 320 µL) was transferred to another Reacti-vial and the solvent removed under a stream of nitrogen. This was re-suspended with 400 µL isopropanol and transferred into an amber LC-MS RAM vial for analysis. Serial dilutions were performed on the mixture of NEFAs from 10 ppm down to 0.3125 ppm. Blank sample was tested with and without internal standard (margaric acid) spike. Samples were run in duplicates with a seven-point calibration curve. A blank sample with internal standard only was analysed before the samples were prepared and compared to the internal standard levels in the final samples to adjust for any NEFA volatility. The LC-MS system included the HPLC Agilent 1200 series connected to the Agilent 6420 triple quadrupole MS/MS. A VisionHT C18, 3 µm, 2.1X50 mm column (Grace Davidson, Auckland, NZ) with guard column (Phenomenex, Auckland, NZ) was used to achieve separation. Column temperature was set at 30°C and the autosampler was set at 8°C. Solvent A was 5 mM Ammonium Formate, 98% Water, 2% ACN and Solvent B was 98% Acetonitrile and 2% Water. At a flow rate of 0.4 mL/min, an Isocratic hold of 87% solvent B was run from 0-3 minutes, and then a gradient of 87%-97% solvent B from 3-7 minutes, and further isocratic hold of 97% solvent B from 7-9 minutes, and finally gradient from 97% back to 87% from 9-12 minutes. The MS settings were: atmospheric pressure chemical ionization mode, negative mode ionization, gas temperature 325°C, gas flow 12 L/min, nebulizer pressure 45 PSI, corona current 8 µA, charging voltage 2000 V, negative voltage 3500 V. Needle injection volume was 5 µL each time with ACN needle wash after each injection. Fragmentor voltage was 100 V for all NEFAs, and Delta EMV was 200 V. Dwell time was 50 milliseconds. MS1 set to wide and MS2 set to unit. Fragmentor voltage was 120 V and collision energy was 20 for all. However, the NEFA did not fragment into daughter ions even at maximum fragmentor voltage and collision energy. Therefore only the MS2 single ion monitoring (SIM) data of the parent ion is taken. The MS2 SIM was set at 253, 255, 269, 279, 281,283 corresponding to palmitoleic, palmitic, margaric, linoleic, oleic and stearic acid, respectively.
Insulin resistance and sensitivity models
Homeostatic model assessment-insulin resistance (HOMA-IR), was calculated [22] as below:
HOMA‑IR: (I0×G0)/22.5, where I0, fasting insulin and G0, fasting glucose.
Matsuda index of insulin sensitivity was calculated from plasma glucose (G0, mg/dl) and insulin (I0, mIU/l) as follow to find out wholebody insulin sensitivity from the OGTT[23] :
In addition, Stumvoll index which is used to predict insulin sensitivity was calculated through insulin baseline and 120min (I0 and I120, pmol/l, respectively) and glucose 120min (G120, mmol/l) as follows [24]:
Stumvoll index = (0.156-0.0000459 I120-0.000321 I0-0.00541G120)
References:
22.Matthews D, Hosker J, Rudenski A, Naylor B, Treacher D, Turner R. Homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28(7): 412-419.
23.Matsuda M, DeFronzo RA. Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes care 1999; 22(9): 1462-1470.
24.Stumvoll M, Gerich J. Clinical features of insulin resistance and beta cell dysfunction and the relationship to type 2 diabetes. Clinics in laboratory medicine 2001; 21(1): 31-51.