Comparison of Tissue Harvest Protocols for the Quantitation of Acylcarnitines in Mouse Heart and Liver by Mass Spectrometry (Supplementary Information)
Christopher Petucci, Stella Rojas-Betancourt, and Stephen J. Gardell
Sanford-Burnham Medical Research Institute, 6400 Sanger Rd., Orlando, FL 32827
Corresponding author: Christopher Petucci, 407-745-2129,
email:
SupplementaryTable 1. Measured acylcarnitines are grouped according to A) acetylcarnitine B) aliphatic saturated acylcarnitines C) aliphatic unsaturated acylcarnitines D) miscellaneous hydroxylated and dicarboxylicacylcarnitines.
GROUP A / GROUP C / GROUP DC2 / C5:1 / C18:2 / C4-OH / C14:1-OH/C12:1-DC
GROUP B / C8:1 / C18:1 / C5-OH/C3-DC / C14-OH/C12-DC
C3 / C10:3 / C20:4 / C4-DC/Ci4-DC / C16:3-OH/C14:3-DC
C4/Ci4 / C10:2 / C20:3 / C5-DC / C16:2-OH/C14:2-DC
C5 / C10:1 / C20:2 / C8:1-OH/C6:1-DC / C16:1-OH/C14:1-DC
C6 / C12:2 / C20:1 / C6-DC / C16-OH/C14-DC
C8 / C12:1 / C22:5 / C7-DC / C18:3-OH/C16:3-DC
C10 / C14:3 / C22:4 / C8:1-DC / C18:2-OH/C16:2-DC
C12 / C14:2 / C22:3 / C10-OH/C8-DC / C18:1-OH/C16:1-DC
C14 / C14:1 / C22:2 / C12:2-OH/C10:2-DC / C18-OH/C16-DC
C16 / C16:3 / C22:1 / C12:1-OH/C10:1-DC / C20:3-OH/C18:3-DC
C18 / C16:2 / C12-OH/C10-DC / C20:2-OH/C18:2-DC
C20 / C16:1 / C14:3-OH/C12:3-DC / C20:1-OH/C18:1-DC
C22 / C18:3 / C14:2-OH/C12:2-DC / C20-OH/C18-DC/C22:6
Supplementary Table 2. Female C57/BL6 mice (regular chow, ad libitum, age as indicated) were anesthetized/sedated/euthanized (A/S/E) according to one of 5 different strategies as indicated. The times between initiation of A/S/E and achieving the desired effect (A/S/E time) and the subsequent time to harvest the tissues are shown. Heart and liver were harvested as soon as possible (ASAP).
A/S/E Strategy / N / Age / A/S/E Time (ave) / Tissue Harvest Time (ave)Ketamine/Xylazine / 5 / 14-15 week / 3.3 min / 53 sec
5 / 14 Week / 2.7 min / 50 sec
Beuthanasia / 5 / 14 Week / 3.3 min / 59 sec
5 / 13 Week / 3.0 min / 46 sec
CO2/CD / 5 / 14 Week / 1.1 min / 54 sec
5 / 16 Week / 1.5 min / 66 sec
Isoflurane / 5 / 14 Week / 1.4 min / 60 sec
5 / 13 Week / 2.8 min / 49 sec
Nembutal / 5 / 14 Week / 4.0 min / 54 sec
5 / 14 Week / 4.9 min / 59 sec
Supplementary Fig. 1: Recovery of deuteratedacylcarnitine internal standards from liver homogenates in 80% methanol compared to water.
Supplementary Fig. 2: Impact of different A/S/E strategies on ACs in mouse heart. Mice were subjectedto different A/S/E strategies: (A) Nembutal (100 mpk, i.p.); (B) CO2/CD; (C) Isoflurane; (D)Ketamine/Xylazine (87/13 mpk, i.p.); and (E) Beuthanasia (75 mpk, i.p.). Hearts were harvested immediately after A/S/E was achieved. Tissues were immediately frozen. Pre-weighed frozen tissues were homogenized directly in 80% methanol and treated as described in the Methods section and assayed for individual ACs. Data is presented as mean (SD); n=10.
Supplementary Figure 3: Impact of different A/S/E strategies on ACs in mouse liver. Mice were subjectedto different A/S/E strategies: (A) Nembutal (100 mpk, i.p.); (B) CO2/CD; (C) Isoflurane; (D)Ketamine/Xylazine (87/13 mpk, i.p.); and (E) Beuthanasia (75 mpk, i.p.). Livers were harvested immediately after A/S/E was achieved. Tissues were immediately frozen. Pre-weighed frozen tissues were homogenized directly in 80% methanol and treated as described in the Methods section and assayed for individual ACs. Data is presented as mean (SD); n=10.
Supplementary Figure 4: Impact of different A/S/E strategies on pooled ACs in mouse heart and liver. Data shown in Figs 1 and 2 (heart and liver, respectively) was used to derive the amount of (A) C2 in the heart and liver, (B) Group B (aliphatic saturated ACs), Group C (aliphatic unsaturated ACs) and Group D (miscellaneous ACs: AC-OH and AC-DC species) in heart and (C) Group B, C, and D in liver. The depicted values show the average of the summation of all grouped ACs (mean; SD; n=10) for each of the 5 treatment groups. Statistically significant differences (relative to the Nembutal treatment group) are indicated. Values that are statistically different (P<0.05) are indicated by an asterisk.
Supplementary Fig. 5: Impact of delayed harvest on the AC levels in heart and liver. The “ASAP Harvest” (from Figures 1 and 2) and “Delayed Harvest” data are plotted adjacent to one another for each measured AC. (A): Heart ACs; (B) Liver ACs. Each data entry represents mean, SD, n=15 (ASAP), n = 14 (delayed harvest, 10 min). Values that are statistically different (P<0.05) are indicated by an asterisk.