Supplementary Information
Carbamazepine inhibits angiotensin I-converting enzyme, linking it to
the pathogenesis of temporal lobe epilepsy
Sandro S Almeida, et al.
Supplementary Data
Supplementary Figure 1: Inhibition of ACE activity by carbamazepine in
ACE-transfected
CHO
cells.
(a)
Percentage
of
inhibition
ACE
by
carbamazepine, represented by percentage of Ang I basal degradation per
min per mg total protein content (HPLC assay). (b) Representative HPLC
elution profile for hydrolysis of Ang I in ACE-transfected CHO cell cultures.
Cells were incubated for 2 h with several concentrations of carbamazepine
(0.05, 0.25, 0.5 and 1.25 mM). Peaks represent Ang I (solid arrows) and AngII peptides (discontinuous arrows), after 30 min of incubation of CHO-ACE
cells with 10 µM Ang I. ACE inhibitor, lisinopril 2.5 µM, was used as control in
the assays (data not show). In the upper figure (zero time of incubation), only
the Ang I peak is shown. In the figure in the middle, the peaks of Ang I and II
have been detected, demonstrating Ang I conversion by ACE in CHO-ACE
cells; in the lower figure, incubation with carbamazepine leads to a smaller
Ang II peak. (c) Mean (± s.e.m.) ACE activity in supernatants of a stably CHO
transfected cell-line inhibited by carbamazepine. CHO cells were previously
transfected with the human somatic ACE form.1 Samples were incubated for 2
h in several concentrations of carbamazepine (0.05, 0.25, 0.5 mM) and
submitted to an enzymatic assay with 10 µM FRET specific ACE substrate
(Abz-FRK(Dnp)P-OH).2 The ACE inhibitor lisinopril (2.5 M) was used as a
control in the assays (data not shown). Mean (± s.e.m.) of triplicate
experiments and the values of ACE activity are shown as arbitrary
fluorescence units (AFUs) of substrate hydrolyzed after 6 minutes of
carbamazepine incubation (***P < 0,001).
Supplementary Data
Supplementary Figure 2: Effect of carbamazepine on kallikrein activity. (a,b)
Mean (± s.e.m.) Carbamazepine at concentrations of 0.25, 0.5, and 2 mM and
0.05, 0.25, and 0.5 mM does not affect human plasmatic (a), or purified (b)
kallikrein activity. Soluble kallikrein activity assay was performed by using the
fluorogenic substrate Z-Phe-Arg-AMC3.
Supplementary Data
Supplementary table 1
Experiment
groups
Genotypes of the study groups
Number of
patients
Genotypes
Treatment
Figure 1a-d
control
TLE
368 ♂
72 ♂
80-II, 187-ID, 101-DD
8-II, 31-ID, 33-DD
---carbamazepine
Figure 1e
TLE- Engel 1A
TLE- other outcomes
65 ♂/♀
75 ♂/♀
5/3-II, 14/14-ID, 10/19-DD
9/3-II, 11/27-ID, 8/17-DD
carbamazepine
carbamazepine
Figure 1f,g
control
TLE
127 ♂
43 ♂
32-II, 59-ID, 36-DD
7-II, 21-ID, 15-DD
---carbamazepine
55 ♂
15 ♂
35 ♂
12-II, 25-ID, 18-DD
1-II, 9-ID, 4-DD
4-II, 16-ID, 15-DD
---non-carbamazepine
carbamazepine
Figure 1h
control
TLE
TLE
TLE, temporal lobe epilepsy.
Supplementary Table 2 Blood pressure results for TLE subjects
TLE - CBZ
TLE – non - CBZ
SBP (mmHg)
124,2 ± 1,812
121,5 ± 4,507
DBP (mmHg)
76,00 ± 1,206
79,92 ± 2,954
MAP (mmHg)
91,89 ± 1,320
93,62 ± 3,169
CBZ, carbamazepine; DBP, diastolic blood pressure; MAP, mean arterial
pressure; SBP, systolic blood pressure; TLE, temporal lobe epilepsy.
Supplementary References
1.
Sabatini RA, Bersanetti PA, Farias SL, Juliano L, Juliano MA, Casarini
DE et al. Determination of angiotensin I-converting enzyme activity in
cell culture using fluorescence resonance energy transfer peptides. Anal
Biochem 2007; 363(2): 255-262.
2.
Carmona AK, Schwager SL, Juliano MA, Juliano L, Sturrock ED. A
continuous fluorescence resonance energy transfer angiotensin Iconverting enzyme assay. Nat Protoc 2006; 1(4): 1971-1976.
3.
Almeida SS, Barros CC, Moraes MR, Russo FJ, Haro AS, Rosa TS et al.
Plasma Kallikrein and Angiotensin I-converting enzyme N- and Cterminal domain activities are modulated by the insertion/deletion
polymorphism. Neuropeptides 2010; 44(2): 139-143.