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: cpetucci@sanfordburnham.org
Supplementary Table 1. Measured acylcarnitines are grouped according to A)
acetylcarnitine B) aliphatic saturated acylcarnitines C) aliphatic unsaturated
acylcarnitines D) miscellaneous hydroxylated and dicarboxylic acylcarnitines.
GROUP A
C2
GROUP B
C3
C4/Ci4
C5
C6
C8
C10
C12
C14
C16
C18
C20
C22
GROUP C
C5:1
C18:2
C8:1
C18:1
C10:3
C20:4
C10:2
C20:3
C10:1
C20:2
C12:2
C20:1
C12:1
C22:5
C14:3
C22:4
C14:2
C22:3
C14:1
C22:2
C16:3
C22:1
C16:2
C16:1
C18:3
GROUP D
C4-OH
C14:1-OH/C12:1-DC
C5-OH/C3-DC
C14-OH/C12-DC
C4-DC/Ci4-DC
C16:3-OH/C14:3-DC
C5-DC
C16:2-OH/C14:2-DC
C8:1-OH/C6:1-DC
C16:1-OH/C14:1-DC
C6-DC
C16-OH/C14-DC
C7-DC
C18:3-OH/C16:3-DC
C8:1-DC
C18:2-OH/C16:2-DC
C10-OH/C8-DC
C18:1-OH/C16:1-DC
C12:2-OH/C10:2-DC
C18-OH/C16-DC
C12:1-OH/C10:1-DC
C20:3-OH/C18:3-DC
C12-OH/C10-DC
C20:2-OH/C18:2-DC
C14:3-OH/C12:3-DC
C20:1-OH/C18:1-DC
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).
14-15 week
14 Week
A/S/E Time
(ave)
3.3 min
2.7 min
Tissue Harvest
Time (ave)
53 sec
50 sec
Beuthanasia
5
5
14 Week
13 Week
3.3 min
3.0 min
59 sec
46 sec
CO2/CD
5
5
14 Week
16 Week
1.1 min
1.5 min
54 sec
66 sec
Isoflurane
5
5
14 Week
13 Week
1.4 min
2.8 min
60 sec
49 sec
Nembutal
5
5
14 Week
14 Week
4.0 min
4.9 min
54 sec
59 sec
A/S/E Strategy
N
Age
Ketamine/Xylazine
5
5
Supplementary Fig. 1: Recovery of deuterated acylcarnitine internal standards from
liver homogenates in 80% methanol compared to water.
C C
4/ 3
C
i
C4
C5
C6
C8
C10
C12
C14
C16
C18
C20
C 22
C5:
C 8:1
C10:1
C10:3
C10:2
C12:1
C14:1
C14:2
C16:1
C16:3
C16:2
C18:1
C18:3
C18:2
C20:1
C20:4
C20:3
C20:2
C22:1
C22:5
C22:4
C
2 3
C 5-O C22:2
4
C -D H/C4 2:1
8:
1- C/CC3-OH
O
D
H Ci4-DC
/C 5 C
6: -D
C
C1-DC
C12: C1
12 2- 0- C C67-DC
: O O 8 - C
C C1-OH/CH/ :1-DC
C14: 12 H/C10C8-DC
C14:3-O-OH 10:2-DC
14 2- H / :1 D
: O /C C - C
C C1-OH/C1210-DC
C16: 14 H/C12:3-DC
C16:3-O-OH 12:2-DC
16 2- H / :1 D
: O /C C - C
C C1-OH/C1412-DC
C18: 16 H/C14:3-DC
C18:3-O-OH 14:2-DC
18 2- H / :1 D
: O /C C - C
C C1-OH/C1614-DC
2
0
C C2 :218-H/C16:3-DC
2 0 0 - O 1 :2 D
-O :1-OH H/ 6: -DC
H O /C C11-DC
/C H 1 6 C
18/C 8: -D
-D18:2-DC
C 1- C
/C D
22 C
:6
nmol/mg of tissue
C C3
4/
C
i4
C
5
C
6
C
C 8
1
C0
1
C2
1
C4
1
C6
1
C 8
5
C :1
C 8:1
1
C 0:3
1
C 0:1
1
C 2:1
1
C 4:2
1
C 4:1
1
C 6:2
1
C 6:1
1
C 8:3
1
C 8:2
1
C 8:1
2
C 0:4
2
C 0:3
2
C 0:
C
5- C 20 2
C OH 4 :1
4- / -O
D C H
C 3C
/C D
12
i4 C
C :2
C -DC
12 -O
:1 H C5-D
-O /C
C
C C1 H 10 6-D
14 2 /C :2 C
C :2 O 10 -D
14 -O H :1 C
:1 H /C -D
-O /C 1 C
C C1 H 12 0-D
16 4 /C :2 C
C :2 -O 12 -D
16 -O H :1 C
:1 H /C -D
-O /C 1 C
C C1 H 14 2-D
18 6 /C :2 C
:
C 2 -O 14 -D
18 -O H :1 C
:1 H /C -D
- /C 1 C
C OH 16 4-D
18 /C :2 C
-O 16 -D
H :1 C
/C -D
16 C
-D
C
nmol/mg of tissue
A
0.012
Heart: Nembutal
0.008
0.004
0.000
B
0.03
Heart: CO 2 + CD
0.02
0.01
0.00
0.025
C C3
4/
C
i4
C
5
C
6
C
C8
1
C0
1
C2
1
C4
1
C6
1
C8
2
C 0
5:
C 1
C 8:1
1
C 0:3
1
C 0:1
1
C 2:1
1
C 4:2
1
C 4:1
1
C 6:3
1
C 6:2
1
C 6:1
1
C 8:3
1
C 8:2
1
C 8:1
2
C 0:4
2
C 0:3
2
C 0:2
C
2
5
C -OH C4 0:1
4- / -O
C
D
1
C C3- H
/C D
C 2:2
12 -O
i4 C
C :1 H/ C -D
14 -O C 5 C
:1 H 10 -D
-O /C :2 C
C C1 H/ 10 -D
16 4 C :1 C
C :2 -O 12 -D
16 -O H :1 C
:1 H /C1 -D
-O /C 2 C
C C1 H/ 14 -D
18 6 C :2 C
C :2 -O 14 -D
18 -O H :1 C
:1 H /C1 -D
/
C
20 C -OH C1 4-DC
-O 18 /C 6:2 C
H -O 16 -D
/C H :1 C
18 /C -D
-D 16 C
C -D
/C C
22
:6
nmol/mg of tissue
C C
4/ 3
C
i4
C
5
C
6
C
C8
10
C
1
C2
1
C4
1
C6
1
C8
C 20
5
C :1
C 8:1
10
C :3
1
C 0:1
1
C 2:1
1
C 4:2
1
C 4:1
1
C 6:3
1
C 6:2
1
C 6:1
1
C 8:3
1
C 8:2
1
C 8:1
2
C 0:4
2
C 0:3
2
C 0:2
2
C
C 0:1
5
C -O C 22
4- H 4 :5
C
8: DC /C3-OH
1- /C -D
i
O
H C4- C
C
1 C /C 5 DC
C 2:2 10 6: -D
12 - -O 1- C
:1 OH H C6 DC
C C -OH/C /C -D
14 1 / 10 8- C
C :2 2- C : D
14 - O 10 2- C
:1 OH H/ :1 DC
C C -OH/C C1 -D
16 1 / 1 0- C
C :2 4- C 2: D
16 - O 1 2- C
:1 OH H/ 2:1 DC
C C -O /C C1 -D
18 1 H 1 2- C
C :3 6- /C 4: D
1
2 C
O
C 8:2-O H 14: -D
18 - H /C 1- C
:1 OH/C1 14 DC
C C -OH/C 6: -D
2 1
1 3 C
C C20:2 8-O/C16:2-DC
20 0 -O H 6 -D
-O :1- H /C :1- C
H OH/C 16 DC
/C / 18 18 C1 :2 DC
-D 8: -D
C 1- C
/C D
22 C
:6
nmol/mg of tissue
C
0.025
Heart: Isoflurane
0.020
0.015
0.010
0.005
0.000
D
Heart: Ketamine Xylazine
0.020
0.015
0.010
0.005
0.000
E
nmol/mg of tissue
0.010
Heart: Beuthanasia
0.005
C C
4/ 3
C
i4
C
5
C
6
C
C8
1
C0
1
C2
1
C4
1
C6
1
C 8
5:
C 1
C 8:1
1
C 0:3
1
C 0:1
1
C 4:2
1
C 4:1
1
C 6:2
1
C 6:1
1
C 8:3
1
C 8:2
1
C
C 8:1
5
2
C -O C4 0:4
4- H D /C OH
C 3
C
/C -D
12
i4 C
C :2
12 -O
C -DC
5
:1 H
- / C -D
C C1OH C10 6-DC
1
/
C 4:2 2-O C10:2-DC
14 -O H : C
:1 H /C 1-D
- / 1
C C1OH C12 0-DC
16 4 /C : C
C :2 -O 12 2-D
16 -O H : C
:1 H /C 1-D
- / 1
C C1OH C14 2-DC
18 6 /C : C
C :2 -O 14 2-D
18 -O H : C
:1 H /C 1-D
- / 1
C OH C16 4-DC
18 /C : C
-O 16 2-D
H :1 C
/C -D
16 C
-D
C
0.000
Supplementary Fig. 2: Impact of different A/S/E strategies on ACs in mouse heart. Mice were
subjected to 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. Preweighed 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.
C C3
4/
C
i4
C
5
C
6
C
8
C
10
C
14
C
16
C
1
C 8
5
C :1
10
C :3
10
C :1
12
C :1
14
C :1
16
C :1
18
C :2
C
5- C 18:
C OH 4- 1
4- / O
D C3 H
C C
/C D
8:
i4 C
1O C -D
H 5 C
/C -D
6: C
1C DC
6C D
C C
12 1 C 7- C
0
C :2- -O 8:1 DC
12 O H :1 H/ /C DC
-O C 81
C C1 H/C 0:2 DC
14 2 1 :1 O 0: DC
-O H/ 1C
C 1 H/C C10 DC
16 4 1 :1 -O 2: DC
-O H/ 1C
C 1 H/C C12 DC
18 6 1 C :2- -OH 4:1 DC
18 O / :1 H/ C1 DC
-O C 4H 16: DC
/C 2
16 -D
:1 C
-D
C
nmol/mg of tissue
0.015
0.030
C C3
4/
C
i4
C
5
C
6
C
8
C
10
C
12
C
14
C
16
C
18
C
2
C 2
5:
C 1
8
C :1
10
C :3
10
C :1
12
C :2
12
C :1
14
C :2
14
C :1
16
C :3
16
C :2
16
C :1
18
C :3
18
C :2
18
C :1
20
C :4
20
C :3
20
C :2
20
C :1
22
:4
C
C
5- C 22:
O
C H 4 1
4- / -O
D C3 H
C C
/C D
8:
i4 C
1O C -D
H 5 C
/C 6: DC
1C DC
6D
C C
12 1 C C7- C
:1 0-O 8: DC
-O H 1C C1 H/C /C DC
14 2
8
C :3- -OH 10: -DC
14 O / 1C :2- H/C C1 DC
14 O 1 0:1 H/ 2: DC
-O C 3C C1 H/C12: DC
16 4 1 2C :3- -OH 2: DC
16 O / 1:1 H/ C1 DC
-O C 2C C1 H/C14: DC
18 6 1 3:
C 2- -OH 4: DC
18 O / 1:1 H/ C1 DC
-O C 4
C H/ 16 -DC
18 C :2
-O 16 -D
H :1 C
/C -D
16 C
-D
C
nmol/mg of tissue
A
Liver: Nembutal
0.010
0.005
0.000
B
Liver: CO 2 + CD
0.025
0.020
0.015
0.010
0.005
0.000
C C3
4/
C
i4
C
5
C
6
C
8
C
10
C
12
C
14
C
16
C
1
C 8
5:
C 1
8
C :1
10
C :3
10
C :1
12
C :1
14
C :2
14
C :1
16
C :2
16
C :1
18
C :3
18
C :2
18
C :1
C
20
5
C -OH C4- :4
4- / O
D C3 H
C C
/C D
8:
i4 C
1O C -D
H 5 C
/C -D
6: C
1C DC
6C D
C C
12 1 C 7- C
C :2- 0-O 8:1 DC
12 O H C :1- H/C /C8 DC
16 O 1 :1 H/ 0: DC
-O C 21
C C1 H/C 0:1 DC
18 6 1 :1 O 4: DC
-O H 1
C H/ /C1 -DC
18 C 4
-O 16 -D
H :1 C
/C -D
16 C
-D
C
nmol/mg of tissue
C C3
4/
C
i4
C
5
C
6
C
8
C
10
C
12
C
14
C
16
C
1
C 8
5
C :1
10
C :3
10
C :1
14
C :1
16
C :1
18
C :2
1
C
5- C 8:1
C OH 4 4 - /C O
D 3 H
C -D
C
/C C
8:
i4
1O C -DC
H 5
/C -D
6: C
1C DC
6C DC
C C
12 10 C 7-D
8
:
C 2- O :1 C
1 2 O H -D
:1 H/ /C8 C
-O C1 D
C
C 1 H/C 0:2 C
14 2- 1 -D
:1 OH 0: C
-O / 1C D
C C1 H/C 10 C
16 4- 1 -D
:1 OH 2:1 C
-O / -D
C
C C1 H/C 12 C
18 6 1 -D
:1 OH 4: C
-O / 1C H/ C1 DC
18 C 4-O 16 DC
H :1/C D
16 C
-D
C
nmol/mg of tissue
C
0.004
Liver: Isoflurane
0.003
0.002
0.001
0.000
D
0.008
Liver: Ketamine/Xylazine
0.006
0.004
0.002
0.000
E
nmol/mg of tissue
0.006
Liver: Beuthanasia
0.004
0.002
C C
4/ 3
C
i4
C
5
C
6
C
C8
1
C0
1
C2
1
C4
1
C6
C 18
5
C :1
C 8:1
10
C :3
1
C 0:1
1
C 2:1
1
C 4:2
1
C 4:1
1
C 6:2
1
C 6:1
1
C 8:3
1
C
C 8:2
5C O C 1 8:
4- H 4- 1
D /C O
C
C 3 H
8:
1- /Ci -DC
4
O
H C -DC
/C 5 6: D
1 C
C -DC
C C
612 1
D
C
C :2 0- C 7- C
1 2 -O O 8 D
:1 H H/ :1- C
- / C D
C C OH C1 8- C
1 1 / 0 D
C 4:2 2-O C1 :2- C
14 -O H 0: D
:1 H /C 1- C
D
C C OH/C1 10- C
16 14 /C 2: D
C :2 -O 1 2- C
1 6 -O H 2 D
:1 H /C :1- C
D
C C OH/C1 12- C
1 8 1 6 /C 4 D
C :3 -O 1 :2- C
18 -O H 4: D
C :2 H /C 1- C
1 8 -O / C 1 D
:1 H 1 4- C
- / 6 D
C OH C1 :3- C
18 /C 6: D
-O 1 2- C
H 6:1 DC
/ C -D
16 C
-D
C
0.000
Supplementary Figure 3: Impact of different A/S/E strategies on ACs in mouse liver. Mice
were subjected to different A/S/E strategies: (A) Nembutal (100 mpk, i.p.); (B) CO 2/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. Preweighed 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.
A
Heart
Liver
0.6
C2 (nmol/mg of tissue)
C2 (nmol/mg of tissue)
0.6
*
0.5
0.4
*
*
0.3
0.2
0.1
0.4
0.3
0.2
*
0.1
K
K
N
e
et Beu mb
am th ut
in an al
e / as
Xy ia
l
Is azi
of ne
lu
ra
C ne
O
2 /C
D
0.0
N
em
et Beu b
am th ut
in an al
e / as
Xy ia
l
Is azi
of ne
lu
ra
C ne
O
2 /C
D
0.0
0.5
B
Heart (ASAP Harvest)
nmol/mg of tissue
0.10
*
0.08
0.06
*
0.04
*
0.02
*
*
*
*
*
*
K
B
N
em
et eu bu
am th ta
in an l
e/ as
Xy ia
l
Is azin
of
lu e
ra
C ne
O
2 /C
D
N
e
B m
K
et eu bu
am th ta
in an l
e/ as
Xy ia
l
Is azin
of
lu e
ra
C ne
O
2 /C
D
N
e
B m
K
et eu bu
am th ta
in an l
e/ as
Xy ia
l
Is azin
of
lu e
ra
C ne
O
2 /C
D
0.00
C
Liver (ASAP Harvest)
Group B (Saturated ACs)
Group C (Unsaturated ACs)
Group D (Miscellaneous ACs)
nmol/mg of tissue
0.10
0.08
0.06
0.04
*
0.02
*
*
K
B
N
em
et eu bu
am th ta
in an l
e/ as
Xy ia
l
Is azi
of ne
lu
ra
C ne
O
2 /C
D
N
e
K B m
et eu bu
am th ta
in an l
e/ as
Xy ia
l
Is azi
of ne
lu
ra
C ne
O
2 /C
D
N
e
K Be m b
et
am uth uta
in an l
e/ as
Xy ia
l
Is azi
of ne
lu
ra
C ne
O
2 /C
D
0.00
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.
C
5:
1
C
C
5
C
4
5O
O
H
C H
4- / C
D C 6
C 3/C D
i4 C
C
-D
8:
C
1O C C
H 5 8
/C 6: D C
1C DC
6D
C C
1
C 0:3
10
:1
C
C 1
C
10 C 7- 0
-O 8: DC
H 1-D
C
/C C
12
8D
C :2
C C
12 -O
12
:1 H
:1
/
-O C
C
1
C H 0 1
12 /C :2 2
-O 10 -D
H :1 C
/C -D
10 C
-D
C
C C
14
14
:1
C :2
-O
14
C H
:1
1 4 /C
-O 12 C1
H :1 4
/C -D
12 C
-D
C
C C
16
16
:1
C :2
-O
16
C H
:1
1 6 /C
-O 14 C1
H :1 6
/C -D
14 C
-D
C C
18
C
C :3
18
18
C :2
C :2
18 -O
18
:1 H
:1
-O /C
C H 16 C1
18 /C :2 8
-O 16 -D
H :1 C
/C -D
16 C
-D
C
0.000
*
0.004
*
*
*
*
Liver Acylcarnitines
0.003
0.002
*
** *
i4
0.005
C
C C
4/ 3
C
C i4
5:
1
C C
C
4- 5
5
C -O
O
4- H
H
D /C C
C 3 6
/C -D
i4 C
-D
C C
8:
1
C C
6- 8
D
C C
10
C :3
C
10
1
:
C 2:2
C1
12 -O
:1 H C1 10
-O /C
C H 1 C2:1
12 /C 0 1
-O 1 :2- 2
H 0:1 DC
/C 10 DC
C
14
C DC
C :2
14
14 -O
:1 H C1 :2
C OH/C1 C4:1
14 /C 2 1
-O 1 :2- 4
H 2:1 DC
/C 12 DC
C DC
C
1
16
C 6:3
C :2
1
16 -O
6
:1 H C1 :2
C OH/C1 C6:1
16 /C 4 1
:
-O 1 2- 6
H 4:1 DC
/C 14 DC
C DC
C
1
18
C 8:3
C :2
18
18 -O
:1 H C1 :2
C OH/C1 C8:1
18 /C 6 1
-O 1 :2- 8
H 6:1 DC
/C 16 DC
C
20
C DC
2
-O
C 0:4
H
20
/C
18 C2 :3
-D C 0:
C 20 2
/C :
22 1
:6
nmol/mg of wet tissue
0.015
C
4/
C
3
nmol/mg of wet tissue
A
Heart Acylcarnitines
ASAP
Delayed (10 min)
0.010
*
*
*
*
B
0.025
ASAP
Delayed (10 min)
0.020
0.015
0.010
0.005
0.000
0.001
*
0.000
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.