Supplemental Material to:
Computational Model of Steroidogenesis in Human H295R Cells to Predict Biochemical Response to Endocrine Active Chemicals: Model Development for Metyrapone
Michael S. Breen,1* Miyuki Breen,2,3 Natsuko Terasaki,4 Makoto Yamazaki,4 Rory B. Conolly2
National Exposure Research Laboratory, U.S. Environmental Protection Agency, 109 T.W. Alexander Drive, Mail E205-02, Research Triangle Park, NC 27711, USA
2 National Center for Computational Toxicology, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA
Biomathematics Program, Department of Statistics, North Carolina State University, Raleigh, NC 27695, USA
4 Safety Research Laboratory, Mitsubishi Tanabe Pharma Corporation, Kisarazu, Chiba 292-0818, Japan
Correspondence should be addressed to: Michael S. Breen, Ph.D. U.S. Environmental Protection Agency Office of Research and Development National Exposure Research Laboratory 109 T.W. Alexander Drive, Mail E205-02 Research Triangle Park, NC 27711 tel: 919-541-9409 fax: 919-541-9444 email: breen.michael@epa.gov
1. Steroidogenesis Assay with H295R cells
NCI H295R human adrenocortical carcinoma cells (American Type Culture Collection, Manassas, VA, USA) were grown in 1:1 mixture of Dulbecco’s modified Eagle’s medium (Invitrogen Corporation, Carlsbad, CA, USA) and Ham’s F12 medium (MP Biomedicals Inc, Irvine, CA, USA) containing 15 mM HEPES (Dojindo Laboratories, Kumamoto, Japan), 0.00625 mg/ml insulin (Sigma-Aldrich, Inc., St. Louis, MO, USA), 0.00625 mg/ml transferrin (Sigma-Aldrich, Inc., St. Louis, MO, USA), 30 nmol/L sodium selenite (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 1.25 mg/ml bovine serum albumin (Sigma-Aldrich, Inc., St. Louis, MO, USA), 0.00535 mg/ml linoleic acid (Sigma-Aldrich, Inc., St. Louis, MO, USA), 2.5 % Nu-Serum I (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), 100 U/mL penicillin (Meiji Seika Kaisha, Ltd., Tokyo, Japan) and 100 mg/L streptomycin (Meiji Seika Kaisha Ltd., Tokyo, Japan) at 37°℃ in a 5% CO2 atmosphere. Cells were grown in 225 cm2 flask (Asahi Techno Glass Corporation, Chiba, Japan) to about 80 % confluence, and then split using 0.025% Trypsin (MP Biomedicals Inc, Irvine, California)- 0.02 % EDTA solution (Dojindo Laboratories, Kumamoto, Japan).
The control and MET exposure experiments were conducted in 6-well tissue culture plates (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). A cell suspension of 2 ml containing 6x105 cells was placed in each well. Each experiment had four replicates per time sample. The test plates were then incubated at 37℃ in a 5% CO2 atmosphere. After incubation for 72 hr, the medium was changed to either 2 ml of supplemented medium, which was1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (Invitrogen Corporation, Carlsbad, CA, USA) containing 0.00625 mg/ml insulin, 0.00625 mg/ml transferrin, 30 nmol/L sodium selenite, 1.25 mg/ml bovine serum albumin, 0.00535 mg/ml linoleic acid, 10 % FBS (Invitrogen Corporation, Carlsbad, CA, USA), 100 U/mL penicillin and 100 mg/L streptomycin, with 50 nM of adrenocorticotropin (ACTH, Sigma-Aldrich, Inc., St. Louis, MO, USA), 20 µM of forskolin (Sigma-Aldrich, Inc., St.
Louis, MO, USA), 100 nM of angiotensin II (EMD Chemicals Inc., Darmstadt, Germany) and 0.1% of dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries, Ltd., Osaka, Japan), in the case of the control experiments, or 2 ml of MET-treated supplemented medium. Dilutions of a MET (Sigma-Aldrich, Inc., St. Louis, MO, USA) stock solution were prepared directly in supplemented medium to generate two test concentrations of MET (1 and 10 uM). After changing the medium, the test plates were incubated at 37℃ with a 5% CO2 atmosphere, and the experiments were initiated. At incubation periods of 8, 24, 48, and 72 hr for the control and MET experiments, the medium and cells were separately removed from four replicate wells. The cells were dissolved in 100 ul of distilled water and sonicated to produce a cell lysate.
The concentrations of twelve steroids (PREG, HPREG, DHEA, PROG, HPROG, DIONE, T, DCORTICO, CORTICO, ALDO, DCORT, CORT) in the medium and cell lysate were measured using liquid chromatography/mass spectrometry (LC-MS/MS). The LC-MS/MS systems consisted of a LC10A VP series (Shimadzu, Kyoto, Japan) and API4000 (Applied Biosystems, Foster City, CA, USA.). The steroids were extracted from the medium and cell lysate by ethyl acetate and separated on LC by acetonitrile and formic acid. MS/MS parameters were optimized using multiple reaction monitoring (MRM) mode for every steroids in positive electrospray ionization. The medium and cell lysate volumes were 500 uL and 70 uL, respectively, and LC-MS/MS running time was 17.5 min/sample. The concentrations of two additional steroids (E1, E2) in the medium and cell lysate were measured using enzyme-linked immunosorbent assay (ELISA) with commercial kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The concentration of cholesterol in the medium and cell lysate was measured using a commercial kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) based on cholesterol oxidase method (Allain et al. 1974).
2. Dynamic molecular balance equations in cells and medium
CHOL in cells:
V dCCHOL,cell cell dt IOL,cell (+) = kV med CHOL,med (t)- k2V cell CHOL,cell
t ; CCHOL,cell (0)=1.88x107 nM
PREG in cells:
V dCPREG,cell (t) cell dt ()= k,Vell CHOL,cell (t)-(k3 + kg + k+19) V cell CPREG,cell (t) + k_19V med CPREG,med (t); CPREG,cel (0) =9.45x103 nM
HPREG in cells:
v dCHPREG,cell (t) dt cell CHPREG,cel (0) =0
= k3V Vcell PREG,cell t)-(k4 + k6 + k
)V C cell +24 HPREG, cell (t) + k
24 V med C
HPREG,med (t);
DHEA in cells:
dCDHEA,cell (t) dt
Vcell -DHEA,cell (= kAV cell CHIPREG,cell (t)-(k7 +k+28 )Vcell CDHEA,cell (t)+k_28V med CDHEA,med (t); CDHEA,cel (0) = 3.49x103 nM
PROG in cells:
cell
dt
v dcPROG,cell () = kV cell CPREG,cell (t)-(kg+ k1 + k+20) V cell CPROG,cell (t)+ K.20V med CPROG,med (t);
CPROG,cell (0) = 32.10 nM
HPROG in cells:
Vel CHPROG,cell (1) = kVell CHPREG,cell (t) + kgV cell CPROG,cell (t)- (kg + k12 + K+25) Cell CHPROG,cell (t) + K_25V med CHPROG,med (t);
dt CHPROG,cell (0)=101.83 nM
DIONE in cells:
Vel DIONE,cell () = k,V/ cell CDHEA,cell (t)+kgV/cell CHPROG,cell (t)- (k10 +K13 + K+29) V cell CDIONE,cell (t)+ K_29V med CDIONE, med (t); cell dCDIONE,cell (t) dt
CDIONE,cell (0) =2.33x103 nM
T in cells:
V dCT,cell (t) cell dt = k10V cell CAD,cell (t)-(k14 +k+31 )Vcell CT,cell (t) + k_31V med CT.med (t)
CT,cell (0) = 0
E1 in cells:
V dC El,cell (t) dt cell
= k k13Vcell C DIONE,cell (t)-(k15 + k
30) V
+30
C cell E1,cell t )+ k V 30 med C El,med (t)
CE1,cell (0) = 2.09x103 nM
E2 in cells:
V dCE2,ovy (t) dt =k14V cell CT,cell (t ) + k15V cell CE1,cell (t )-k+32Vcell CE2,cell (t ) + k_32V med CE2.med (t)
cell CE2,cell (0) = 424.17 nM
DCORTICO in cells:
V dCDCORTICO,cel (t) k16 dt = KIV cell CPROG,cell (t)- 16 + K21 VellCDCORTICO,cell (t)+ k-21V med CDCORTICO, med (t
(t)
cell CDCORTICO,cell (0) = 835.01 nM
1
aCORTICO
CORTICO in cells:
V dCCORTICO,cell dt (t) cell = k16
V cell
C DCORTICO,cell (t)-(k1 + k K+2 +22)1
)V
cell C
med CORTICO,cell (t)+k 22 V CORTICO,med (t)
a CORTICO
CCORTICO,cell (0) = 2.27x103 nM
ALDO in cells:
V aCALDO,cell (1) = K V/cell CORTICO,cell (t) - K+23V cell CALDO,cell (t) + k_23V med CALDO,med (t) cell dt
CALDO,cell (0) = 0
DCORT in cells:
V dC DCORT,cell
dt (t) = k12V cell CHPROG,cell (t)-
⎜ ⎛ ⎝ k17 a
+ k
V
C cell DCORT,cell (t) t) + -26
k V. med DCORT,med (t)
cell
CORT
CDCORT,cell (0) = 7.56x104 nM
CORT in cells:
cell
CORT,cell (t) =
V dC dt @CORT
K17_V V cell DCORT,cell t)-k +27
cell
C CORT,cell (t)+k_27 V med 1C CORT,med (t)
CCORT,cel (0) = 3.43x103 nM
CHOL in medium:
dC CHOL,med (t) =
-) k C CHOL,med (t);
CHOL.me (0) =8.11x104 nM
dt
PREG in medium:
V
med dCPREG,med (t) dt
= k +19 V
PREG,med cell C PREG,cell t)-k -19 V med C PREG,med
(t); ( C (0) = 0.85 nM
HPREG in medium:
V dC HPREG,med V med dt (t) = k +24 cell C HPREG,cell t) - k V
HPREG,med 24 med C HPREG,med (t); C (0) = 69.45 nM
DHEA in medium:
V dC C dt DHEA,med (t) = k V +28 cell DHEA,cell t) t)-k k_2 V -28 med
med C
DHEA,med A,med (t); C
DHEA,med (0) = 0
PROG in medium:
+26 ⎞ ⎠ ⎟
V med dCPROG,med (t) PROG,med
= k +20
V
C
cell
PROG,cell t -
k 20 V
led P
PROG,med t); PROG,med
C (0) = 0.03 nM
HPROG in medium:
V dC HPROG,med dt (t) k V +25 med
cell C HPROG,cell t )-k
-25
V C med HPROG,med (t);
C HPROG,med (0)= 0
DIONE in medium:
V dC DIONE,med dt (t) med =
k K ,291 V
cell C DIONE,cell t)
-1 k
V
29 med C
(t);
DIONE,med ( DIONE,n DIONE,med (0) = 0.80 nM
T in medium:
V T,med dC. dt (t) = k 1Vcell CT,cell (t)-k_31Vmed CT,med (t); CT,med (0) = 0.80 nM med +31
E1 in medium:
V
med dC
E1,med dt (t) = kVenC.
E1,cell t) - k
30 V
med C E1,med (t);
C E1,med (0) = 0.11 nM
E2 in medium:
V
med dC E2,med dt (t) = k V +32 cell C E2,cell
t )- k V 32 med E2,med t ; CE2
E2,med (0)=1.21 nM
DCORTICO in medium:
V
med dC DCORTICO,med dt (t) =
k +21Vo
cell C DCORTICO,cell (t) )-k 21 V n C
med DCORTICO,med (t);
DCORTICO,med (0) = 0 nM
CORTICO in medium:
V.
med dC CORTICO,med dt (t) =
k +22
V
cell C CORTICO,cell (t) )-1 k -22
V. med C CORTICO,med (t); CORTICO,med 1 C (0) = 0.11 nM
ALDO in medium:
dt
V dC ALDO,med
med
k V dt (t) +23 cell ALDO,cell C
(t)
V -k 23 3 med ALDO, med C
(t); C ALDO,med (0) = 0.91 nM
DCORT in medium:
V
dc DCORT, med () = K Vel CDCORT. cell (t) -k_ 26 med CDCORT, med (t); CDCORT, med (0) = 0 nl k 26V cell - (t ); med dCDCORT,med (t) dt
CORT in medium:
V dCCORT,med (t)_1 V C
med
CORT,med dt
k +2 +27
cell CORT,cell (t) - k
V
27 med C
CORT,med (t); C CORT,med (0) = 0.03 nM
3. Molecular balance equations for quasi-equilibrium
CHOL in cells:
dC
V cell
CHOL,cell dt (t)
1 = kV med CHOL, med (t)- k2V cell CHOL,cell (t); CHOL,cell (0) = 1.88x107 nM
PREG in cells:
dC
dt CPREG,cell (t)
= ⎝
V
cell
+ V 1 med 919 ⎠ k2V cell C
CHOL,cell t)- k3 + k5 )
cell CH
PREG,cell (t)];
CPREG,cell (0) = 9.45x103 nM
HPREG in cells:
dC HPREG,cell dt (t)
= ⎝ V
+ 1 V
cell
med 924 HPREG,cell İL k3V cell C PREG,cell t) k4 + k 6 )V cell HPREG,cell (t)]; C (0) = 0
DHEA in cells:
dC DHEA,cell (t)
q28 28 JC
dt V. KAV Cell CHPREG cell (t)-KV cell CDHEA,cell (t)]; CDHEA,cell (0) = 3.49x103 nM 1 = + V med 1 cell
PROG in cells:
dCPROG,cell (t) dt
= ⎝ V cell
+ V.
med 920 IL k5V cell C
PREG,cell t)-(kg + ku )
cell C
PROG,cell (t)];
, PROG,cell
(0) = 32.10 nM
HPROG in cells:
dC HPROG,cell dt (t)
= ⎝ Vcell + V 1
⎞ ⎠ k;V
C HPREG,cell (t)+ kgV cell
C PROG,cell t
-(k (kg + k12 )V IC cellHPROG,cell (t)]
CHPROG,cell (0) =101.83 nM
DIONE in cells: dC DIONE,cell dt (t) =
⎜ ⎛ V
1
⎞
⎝
cell
med 929
⎠
k,V cell CDHEA,cell (t) + kgV cell CHIPROG,cell (t)-(k10 +k13 )Vcell CDIONE,cell (t)]
CDIONE,cell (0) =2.33x103 nM
T in cells:
dCT,cell (t) dt
= ⎝ V
1
med 931 ⎠ [KroV cell CDIONE,cell (t)-k14/ cell CT,cell (t)]; CT,cel (0) = 0
E1 in cells:
dC
E1,cell dt t)
= ⎝ V cell
930
+ V 1 med JE k13Vcell CDIONE,cell (t)-k15/cell CE1,cell (t)]; CE1,cell (0) = 2.09x103 nM
E2 in cells:
dC
E2,ovy dt
(t)
= ⎝ V cell
V + 1 med 932
IL LK14V cell CT,cell (t)+ k15V/cell CE1,cell (t)]; CE2,cel (0) = 424.17 nM
+ V
cell
+ V
[
cell
med 925
1
DCORTICO in cells:
dC ‘DCORTICO,cell dt (t)
V ⎜ ⎛ ⎝ = cell
+ 1 V med 921 kyV
DI
C cellPROG,cell (t) -
a
K16 CORTICO V cell
C DCORTICO,cell (t) ‘DCORTICO,cell ⎥ ; C (0) = 835.01 nM
CORTICO in cells:
dC CORTICO,cell dt
(t) 1 Vceu + Vmed 922 JL ⎛
DI
k16 CORTICO V
a
C cell DCORTICO,cell (t)
- k18
cell
C ‘CORTICO,cell CORTICO,cell (t) ⎥ ; C (0) = 2.27x103 nM
ALDO in cells:
dC ALDO,cell
dt (t)
= ⎝ V
+ 1 V
cell
med 923 IL [KigVcel CCORTICO,cell (t)]; CALDO,cel (0) = 0
DCORT in cells:
dC DCORT,cell
dt (t)
= ⎝ V
+ 1 V med DI
926 K12V cell C HPROG,cell (t)-
a
k17 C CORT V cell ‘DCORT,cell (t) ⎥
; CDCORT,cell (0) = 7.56x104 nM
CORT in cells:
dC CORT,cell (t)
dt
= ⎝ V
1 + V
927 )
k17 CORT V cell
C DCORT,cell (t) ⎥ ;
CORT,cell
C (0) = 3.43x103 nM
cell
med
a
cell
FIGURE LEGENDS
Supplemental Material, Figure 1. Graphical representation of the parameters for the mathematical H295R steroidogenesis model. First-order rate constant for cholesterol uptake into the cells is k1 . First-order rate constants for metabolic processes are: k2 - k18 . Reversible first-order rate constants for transport processes (k+x and k_x for secretion and import of steroid x; respectively) are
k19 - k32 . Enzyme inhibition constants for MET are k41 and k42 for CORTICO and CORT pathways, respectively.
Supplemental Material, Figure 2. Comparison of transport equilibrium model-predictions (linear regression line) with measurements in cells and medium. Model-predicted DCORTICO concentrations in medium were plotted as a function of DCORTICO concentrations in cells, and compared with mean concentrations measured at five sampling times for control and two MET concentrations.
Supplemental Material, Figure 3. Model evaluation of transport pathway. Comparison of transport equilibrium model-predictions with time-course measurements in medium from control (a) and two MET concentrations: 1 uM (b) and 10 uM (c). Model-predicted and mean measured DCORTICO concentrations in medium were plotted at five time points after incubation of cells with MET. Model-predicted DCORTICO concentrations in medium were estimated from mean measured concentrations in cells at each corresponding time point. Dotted lines represent linear interpolations between model-predicted and measured concentrations. Measured steroid concentrations are same as shown in Fig. SF2.
Supplemental Material, Figure 4. Model evaluation of metabolic pathway for control experiments. Model-predicted concentrations in cells were plotted as a function of time, and compared with concentrations (mean and standard deviation) measured at five sampling times for steroids: ALDO, E2, T (a); PROG, HPROG, DHEA (b); HPREG, DIONE, E1 (c); CORTICO, DCORTICO (d); PREG, CORT, DCORT (e).
Supplemental Material, Figure 5. Model evaluation of metabolic pathway for control and MET-exposed cells. Model-predicted concentrations in cells were plotted as a function of time, and compared with concentrations (mean and standard deviation) measured at five sampling times for steroids: ALDO (a), CORTICO (b), CORT (c), DCORTICO (d), DCORT (e). For controls, model- predicted and measured steroid concentrations are same as shown in Fig. SF4.
K2
H295R Cells
CHOL
K3
KĄ
PREG
HPREG
DHEA
K5
K6
K7
PROG
kg
HPROG
Kg
K10
DIONE
T
K11
K12
K13
K14
k
DCORTICO
15
DCORT
E1
E2
K16
K41
MET
k <42
K17
CORTICO
CORT
k 18
ALDO
k1
K19 ₭20 k21
K2
K23
k24
940 K25
k26
k27
k28
k29
K30
K31
K32
CHOL
MET
PROG
CORTICO
HPREG
DCORT
DHEA
E1
E2
PREG
DCORTICO
ALDO
HPROG
CORT
DIONE
T
Medium
7000
-Model-predicted DCORTICO
Concentration in Medium (nM)
6000
Measured DCORTICO: Control
Measured DCORTICO: MET=1uM
Measured DCORTICO: MET=10uM
5000
4000
3000
2000
1000
0
0
0.5
1
1.5
2
2.5
3
3.5
4
Concentration in Cells (nM)
4.5 5
× 10
A
7000
. Measured DCORTICO: Control
Concentration in Medium (nM)
6000
Model-Predicted DCORTICO: Control
5000
4000
3000
2000
1000
10
20
30
40
50
60
70
80
Time (hr)
B
7000
I.Measured DCORTICO: MET=1uM
Concentration in Medium (nM)
6000
Model-Predicted DCORTICO: MET=1uM
5000
4000
3000
2000
1000
10
20
30
40
50
60
70
80
Time (hr)
C
7000
A.Measured DCORTICO: MET=10uM
Concentration in Medium (nM)
6000
A. Model-Predicted DCORTICO: MET=10uM
5000
4000
3000
2000
1000
0
10
20
30
40
50
60
70
80
Time (hr)
A
9000
-Model-Predicted ALDO
Measured ALDO
Concentration in Cells (nM)
7500
Model-Predicted E2
Measured E2
6000
Model-Predicted T
Measured T
4500
3000
1500
K
Ű
10
20
30
40
Time (hr)
50
60
70
80
B
C
x 10
4
10
4
Model-Predicted PROG
10
× 10
Measured PROG
Model-Predicted HPREG
Measured HPREG
Concentration in Cells (nM)
8
Model-Predicted HPROG
Measured HPROG
Concentration in Cells (nM)
8
Model-Predicted DIONE
Model-Predicted DHEA
Measured DIONE
Measured DHEA
Model-Predicted E1
I
Measured E1
6
6
4
4
2
2
0
10
20
30
40
50
Time (hr)
60
70
80
0
10
20
30
40
50
60
70
80
Time (hr)
D
E
x 10
5
8
8
10
5
Model-Predicted CORTICO
Model-Predicted PREG
Measured CORTICO
Measured PREG
Concentration in Cells (nM)
Model-Predicted DCORTICO
Model-Predicted CORT
6
Measured DCORTICO
Concentration in Cells (nM)
6
Measured CORT
Model-Predicted DCORT
Measured DCORT
4
4
2
2
10
20
30
40
Time (hr)
50
60
70
80
0
10
20
30
40
Time (hr)
50
60
70
80
A
10000
Model-Predicted ALDO: Control
Measured ALDO: Control
Concentration in Cells (nM)
Model-Predicted ALDO: MET 1uM
7500
Measured ALDO: MET 1uM
-Model-Predicted ALDO: MET 10uM
Measured ALDO: MET 10uM
5000
2500
0
10
20
30
40
Time (hr)
50
60
70
80
B
C
3
x 10
5
4
Model-Predicted CORTICO: Control
15
10
Model-Predicted CORT: Control
Measured CORTICO: Control
Measured CORT: Control
Concentration in Cells (nM)
Model-Predicted CORTICO: MET 1uM
Measured CORTICO: MET 1uM
Concentration in Cells (nM)
12
Model-Predicted CORT: MET 1uM
2.25
Measured CORT: MET 1uM
-Model-Predicted CORTICO: MET 10UM
-Model-Predicted CORT: MET 10uM
Measured CORTICO: MET 10uM
. Measured CORT: MET 10uM
9
1.5
6
0.75
3
0
10
20
30
40
Time (hr)
50
60
70
80
0
0
10
20
30
40
50
60
70
80
D
E
Time (hr)
x 10
5
10
5
10
Model-Predicted DCORTICO: Control
10
Model-Predicted DCORT: Control
Measured DCORTICO: Control
Measured DCORT: Control
Concentration in Cells (nM)
8
Model-Predicted DCORTICO: MET 1uM
8
Model-Predicted DCORT: MET 1uM
Measured DCORTICO: MET 1uM
Concentration in Cells (nM)
Measured DCORT: MET 1uM
-Model-Predicted DCORTICO: MET 10uM
-Model-Predicted DCORT: MET 10uM
Measured DCORTICO: MET 10UM
Measured DCORT: MET 10uM
6
6
4
4
2
2
d
0
10
20
30
40
50
60
70
80
0
10
20
30
40
Time (hr)
50
60
70
80
Time (hr)
| Steroid | Quantitative range (nM) | |
|---|---|---|
| Cells | Medium | |
| PREG | 1.3x104- 1.3x106 | 15.8 - 1.6x103 |
| HPREG | 1.2×104- 1.2×106 | 15.0 - 1.5x103 |
| DHEA | 1.4x104 - 1.4x106 | 17.3 - 1.7x103 |
| PROG | 2.6×103- 1.3×106 | 3.2 - 1.6x103 |
| HPROG | 2.4×103- 1.2×106 | 3.0 - 1.5x103 |
| DIONE | 2.8x103- 1.4x106 | 3.5 - 1.7x103 |
| T | 2.8x103-1.4x106 | 3.5 - 1.7x103 |
| E1 | 1.6x103 - 1.5x105 | 5.5 -5.5x102 |
| E2 | 5.0x102- 1.0x104 | 5.5 - 1.1x102 |
| DCORTICO | 1.2×104- 6.1x106 | 15.1 - 7.6x103 |
| CORTICO | 1.2x104 - 5.9×106 | 14.4 -7.2×103 |
| ALDO | 2.2×103-1.1x106 | 2.8 - 1.4x103 |
| DCORT | 1.2×104- 5.9x106 | 14.4 - 7.2×103 |
| CORT | 1.1x104- 5.6x106 | 13.8 - 6.9x103 |
Supplemental Material, Table 1