Analysis of 3-Hydroxyoctanoic Acid (3-OH-C8)

Supplementary Data (Manuscript Ahmed et al.)

Analysis of 3-hydroxyoctanoic acid (3-OH-C8)

3-OH-C8 concentrations in samples from patients treated with ketogenic diet were determined according to Moser et al. with slight modifications. Plasma samples were analyzed after liquid-liquid extraction with n-hexane and methylation using GC/MS in selected-ion-monitoring mode (SIM). In brief, internal standard C19:0 was added to 100 µL plasma. After addition of 1 ml 3 N methanolic HCl the reaction mixture was heated for 1 hour at 80 °C. 2 ml potassium carbonate (14% w/v) was added and the neutralized sample was extracted two times with 2 ml n-hexane. After evaporating the solvent the resulting residue was redissolved in 50 µl dichloromethane. For GC/MS analysis an Agilent 6890 series GC and Agilent 5975A mass selective detector was used with a DB5-MS capillary column (25 m  0.25 mm I.D., film thickness 0.25 µm) obtained from J&W. Helium was used as the carrier gas (linear velocity: 44 cm/s). A total of 1 µl was injected splitless (0.5 min) at 280 °C. Temperature program was as follows: 60 °C (2 min), 14.41°C/min to 160°C (0 min), 5.77 °C/min to 300 °C (4.86 min). The electron energy was set at 70 eV and the ion source temperature was set at 200 °C. 3-OH-C8 was monitored at m/z 103 (quant ion) and m/z 124 (qual ion), each with a dwell time of 50 ms. Three-point calibration curves were acquired for 3-OH-C8 in a range of 0 - 6.2µmol/l and 12.4 - 31 µmol/l using 10 nmol C19:0 as internal standard.

For the determination of 3-OH-C8 levels in plasma samples of patients with diabetic ketosis, stable isotope-labeled 3-OH-octanoate (3-OH-C8) was synthesized and used to quantify the amount of native 3-OH-C8 in the samples as previously described (Jones et al., 2000). Briefly, 10 µl of 500 µmol/l stable isotope-labeled 3-OH-C8 was added to 500 µl of sample. Samples were acidified with 125 µl of 6 M HCl and then extracted twice with 3 ml ethyl acetate each time. The combined ethyl acetate extractions were dried with sodium sulfate and then dried down at 37º C under a stream of nitrogen. Samples were then derivatized with 100 µl of BSTFA + TMCS (99:1) [N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchlorosilane (BSTFA + TMCS, 99:1) from Supelco] at 75 ºC for 0.5 to 2 hours. 1 l was used for analysis by GC/MS using a Hewlett-Packard 5890 Series II gas chromatograph with a 5972 Series quadrupole mass spectrometer. A split/splitless injector in splitless mode at 270C introduced the sample onto a Hewlett-Packard HP-5MS capillary column [30 m X 0.25 mm(i.d.)] coated with a 0.25 m film of cross-linked 5% PH ME Siloxane. GC temperature parameters were: 80 C for 5 min, ramp 3.8 C/min to 140 C, ramp 2.3 C/min to 200 C, and then ramp 15.0 C/min to 290 C and hold for 6 minutes. The column is inserted directly into the ion source at an interface temperature of 290 C. This ion source utilizes electron impact ionization. Native and stable isotope-labeled 3-OH-C8 were detected by selected ion monitoring (SIM) for the [M -CH3]+ fragments from each, (m/z 289 and 291, respectively). A dwell time of 50 ms was used. Native 3-OH-C8 was quantified by calculating the ratio of native to stable-isotope present and multiplying by the concentration of stable-isotope.

Analysis of 3-hydroxybutyrate (3-HB)

For quantitative determination of 3-OH-butyrate in plasma of patients receiving ketogenic diet, the Autokit 3-HB from Wako Chemicals (Neuss, Germany) was chosen which is designed to be used on an Olympus AU400 Analyzer (Olympus GmbH, Hamburg, Germany).

3-OH-butyrate levels in plasma samples of patients with diabetic ketosis were quantified by using the Stanbio β-Hydroxybutyrate LiquiColor® Procedure No. 2440 kit from Stanbio Laboratory, Boerne, Texas. The kit was adapted for use on a Cobas FARA semi-automated instrument for this analysis.

References for Supplementary data

Moser HW, Moser AB (1991) “Measurements of saturated very long chain fatty acids in plasma”. In Hommes FA, ed. Techniques in Diagnostic Human Biochemical Genetics. New York: Wiley-Liss, 177-191.

Jones PM, Quinn R, Fennessey PV, Tjoa S, Goodman SI, Fiore S, Burlina AB, Rinaldo P, Boriack RL, Bennett MJ. Improved stable isotope dilution-gas chromatography-mass spectrometry method for serum or plasma free 3-hydroxy-fatty acids and its utility for the study of disorders of mitochondrial fatty acid -oxidation. Clin Chem 46(2):149-55. 2000.