Changes in Acetyl CoA Levels during the Early Embryonic Development of Xenopus laevis
Figure 1
Chromatographic separation of CoA standards and a PCA extract of Xenopus embryos.
(A) HPLC chromatogram illustrating separation of CoA compounds. Standards of CoASH, CoA thioesters and adenosine (20–50 pmol each) were separated on a C18 column (Kinetex C18 100 X 4.60 mm column with 2.6 µm particle size and 100 Å pore size) using a mobile phase consisting of 150 mM Na2H2PO4 and 9% methanol and a flow rate of 0.8 ml/min (see Materials and Methods for details). Standards were made up in mobile phase, which additionally contained 5 mM EDTA and 10 mM TCEP before injection. CoA compounds were detected by absorbance at 254 nm. Retention times (in minutes): malonyl CoA, 3.21; adenosine, 4.16; CoASH, 5.06; methylmalonyl CoA, 7.26; dephospho CoA, 10.7; succinyl CoA, 11.87; HMG/acetoacetyl CoA, 13.96; acetyl CoA, 14.83. See Table S1 for day-to-day variability in retention times. (B) Graphs showing linearity between the peak area and the amount of CoASH and acetyl CoA injected. Different amounts of CoASH and acetyl CoA standards were injected and the peak areas were determined by Borwin chromatography software. Each data point represents the mean of duplicate measurements. The limit of detection (LOD) for each compound was determined to be 5 pmol (100 nM). (C) Representative chromatogram showing separation of a PCA extract of stage 8/9 Xenopus embryos. CoA peaks were identified by comparison of retention times with those of authentic standards determined on the same day. Peaks corresponding to CoASH, succinyl CoA, HMG/acetoacetyl CoA and acetyl CoA were detected. Retention times (in minutes): CoASH, 5.21; succinyl CoA, 12.32; HMG/acetoacetyl CoA, 14.44; acetyl CoA, 15.43.
