Wei et al, SI

Supplementary Methods

Recombinant Protein Expression and Purification

The 6xHis-ERRα-LBD plasmid reported by Greshik et al (Greschik et al., 2008) was provided

courtesy of Dino Moras. 6xHis-ERRα-LBD was expressed in Rosetta 2 (DE3) cells. Terrific

broth was inoculated with a 1:50 dilution of starter culture. Cells were grown to OD 0.4 at 37°C

and then moved to 18°C. Once they reached OD 0.8 they were induced with 0.1mM IPTG and

grown for 16 hours before harvesting. 6xHis-ERRα-LBD was purified by nickel affinity

chromatography followed by size exclusion chromatography (SEC).

Total Lipid Extraction from Mouse Tissues

Lipid extraction was performed based on known protocol (Bligh and Dyer, 1959; Saghatelian et

al., 2004). Brain (200 mg) was dounce homogenized on ice for 40 strokes in a mixture of 1 mL:

1 mL: 2 mL water/methanol/chloroform. The resulting mixture was centrifuged at 1000 x g for 10

minutes at 4 ºC to separate organic and aqueous phases. The organic phase containing

extracted lipids (the lipidome) was transferred to an empty pre-tared vial using a Pasteur

pipette. This sample was then dried under a gentle stream of N2 and stored at -80 ºC, if needed.

Lipids were extracted from kidney (200 mg) using a similar protocol. Dried extracts were

weighed and then dissolved in DMSO (40 μl/mg) and used for subsequent experiments.

Pull-Down of Global Lipids from Tissue Extraction

Centrifuge columns (Pierce #89868) were loaded with 10μl IMAC sepharose 6 Fast Flow beads

(GE). Beads were washed with water (2 x 500μl) and the wash was removed by centrifugation.

100μl 0.2M nickel sulfate was added to each column and the column was incubated for 30 min.

Nickel sulfate was then eluted, and columns were washed with water (3 x 100μl) and protein

buffer (20mM Tris, 200mM NaCl, pH 8.0) (3 x 200μl). 200μl of 25μM or 50μM HIS-ERRα-LBD

1
 Wei et al, SI

or protein buffer containing no protein was incubated with resin for 2 hours. After incubation,

protein was eluted and beads were washed with protein buffer containing 50mM imidazole (3 x

200μl). To prepare the lipid mixture, brain lipid extracts were dissolved in DMSO at 40μl/mg,

vortexed, and then sonicated in a sonicator bath to ensure solubilization of the lipids. 12% lipid

mixture was prepared by adding the DMSO lipid stock to protein buffer at 12% by volume. This

mixture was vortexed and sonicated in a sonicator bath, and then 100μl was added to column

and incubated for 30 min. The lipid mixture was then eluted and columns were washed with

protein buffer containing 50mM imidazole (3 x 100μl). Protein-lipid conjugate was then eluted

with protein buffer containing 250mM imidazole (3 x 100μl), and 80μl were injected on Agilent

6220 LC-MS TOF in negative or positive mode. For mass spectrometry, capillary voltage was

3500 V, fragmentor voltage was 100 V; nebulizer gas was 45 psi; drying gas temp was 350°C

with a flow of 10 L/min; m/z range was from 100-1500 Daltons. This procedure was done in

triplicate.

LC Gradient of Untargeted Lipidomics Method

An Agilent LC 1200 was used with a C4 biobond 5µm, 50 x 4.6 mm column

Solvent A: 95% water, 5% methanol

Solvent B: 60% isopropanol, 35% methanol, 5% water

Positive mode solvent modifier: 0.1% formic acid and 5mM ammonium formate

The gradient began with 0% solvent B for the first 12 minutes. From minute 12 to minute 52

solvent B increased from 0% to 100%. The gradient was held at 100% B until minute 58. B was

then decreased to 0% until minute 65 in order to equilibrate the column. The flow rate was 0.5

ml/minute.

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 Wei et al, SI

Data Analysis of Untargeted Lipidomics

After LC-MS TOF analysis, data from replicates was aligned using XCMS. Data was filtered for

ions with greater than 100,000 average counts (area under the curve) in either the protein or

resin samples. Ions that were not present in the lipid sample alone were also removed. To

identify ions of interest, data was further filtered for p < 0.05 (1 tailed t-test) and fold change > 2.

Sterol Isolation from Mouse Tissues and Cells

For tissue samples, total lipids were isolated in the same manner as described above, except

that the extracts were dissolved in 1 ml toluene after drying under nitrogen. Sterols were then

fractionated from total lipids using solid phase extraction (Silica cartridge, 100 mg/1 ml,

BJ9050). The column was equilibrated with 1 ml hexanes, loaded with the lipid sample, washed

with 1 ml hexanes, and then sterols were eluted with 30% isopropanol in hexanes (8 ml). After

elution, the sample was dried under nitrogen, weighed, and dissolved in DMSO or ethanol at 40

μl/mg, then 960 μl protein buffer (20mM Tris, 200mM NaCl, pH 8.0) per mg was added to the

DMSO/ethanol stock, to make the ratio of organic solvent volume to total solvent volume equal

to 4%, and 20μl of this lipid solution was injected on Agilent 6410 LC-MS QQQ to confirm that

sterols were dissolved. In all cases, cholesterol ion intensity was greater than 200,000 counts.

For cell samples, 1 ml PBS, 1 ml methanol and 2 ml chloroform was added to extract total lipids.

Cell samples were then shaken and vortexed to homogeneity. After centrifugation for 6 min at

2200 g, the organic layer (chloroform/methanol) was collected and dried under nitrogen. The

dried extract was dissolved in 1 ml toluene, and the sterols were then fractionated using solid

phase extraction in the same manner as tissue sample, except that after elution, the sample

was dried under nitrogen and dissolved in 50 μl methanol, and 10 μl of this lipid sample was

injected on Agilent 6410 LC-MS QQQ for sterols analysis.

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 Wei et al, SI

Pull-Down for the Targeted Identification of Sterols from Tissue

The sterol pull-down was performed as the global lipid pull-down above except a 4% (v/v) sterol

mixture (as described in the sterol isolation section above) was used in place of a lipid mixture.

After elution, aliquots were extracted into chloroform/methanol layer using a chloroform-

methanol-water mixture (2:1:1) and dried down under nitrogen, dissolved in 30μl water, and

then 20μl was injected on Agilent 6140 LC-MS QQQ.

LC-MS MRM Method for Sterols Analysis

Our method to identify sterols was developed from work by Shan et al. and McDonald et al

(McDonald et al., 2007; Shan et al., 2003). Sterols were analyzed on an Agilent 6140 LC-MS

QQQ. A Phenomenex C18 column (100Å, 3µm, 250x2mm) was used for sterols separation.

Solvent A was 85% methanol, 15% water, and 5mM ammonium acetate. Solvent B was 100%

methanol and 5mM ammonium acetate. The linear gradient is shown in the table below.

Time (min) %B Flow rate(mL/min)

0 0 0.25
12 0 0.25
45 100 0.25
65 100 0.25
65.1 0 0.25
78 0 0.25

The MS was operated in positive ion mode with multiple-reaction monitoring (MRM) for sterols

detection. Capillary voltage was set at 4000 V, and the nebulizer gas was set at 35 psi, with

drying gas flow rate of 8 L/min and temperature of 100°C. The detailed mass spectrometric

parameters of MRM method are described in the table below.

4
 Wei et al, SI

Precursor MS1 Product MS2 Dwell Fragmentor Collision

Sterol compound energy (V)
ion (m/z) resolution ion (m/z) resolution time (ms) (V)
Saturated-tetraol 454.4 Wide 367.3 Unit 80 65 15
Unsaturated-tetraol 452.4 Wide 365.3 Unit 80 65 15
Lanosterol 444.4 Wide 409.4 Unit 80 95 15
Saturated-triol_1 438.4 Wide 420.4 Unit 80 65 15
Saturated-triol_2 438.4 Wide 385.3 Unit 80 65 15
Saturated-triol_3 438.4 Wide 367.3 Unit 80 65 15
Unsaturated-triol_1 436.4 Wide 418.4 Unit 80 65 15
Unsaturated-triol_2 436.4 Wide 383.3 Unit 80 65 15
Unsaturated-triol_3 436.4 Wide 365.3 Unit 80 65 15
24-dihydrolanosterol 429.4 Wide 411.4 Unit 80 115 17
OH-Chol /
420.4 Wide 385.3 Unit 80 65 15
Epoxy-Chol_1*

OH-Chol /
420.4 Wide 367.3 Unit 80 65 15
Epoxy-Chol_2
24, 25-Epoxy-Chol 418.4 Wide 383.3 Unit 80 60 12
Cholestanol 406.4 Wide 371.4 Unit 80 125 13
Cholesterol /
404.4 Wide 369.4 Unit 80 125 13
Lathosterol

Desmosterol /
402.4 Wide 367.3 Unit 80 70 20
20α-OH- Chol
7-keto-Chol 401.3 Wide 383.3 Unit 80 95 35
Unsaturated-Chol 385.3 Wide 367.3 Unit 80 100 15
7α-OH- / 7β-OH-Chol /
385.3 Wide 367.3 Unit 80 65 15
Cholestenone

* Chol refers to cholesterol, OH-Chol refers to hydroxy derivatives of cholesterol, and Epoxy-Chol refers to epoxy
derivatives of cholesterol.

Diethylstilbestrol (DES) enrichment

The lipid mixture was spiked with 79 μM DES, and the global lipid pull-down procedure above

was repeated with this mixture. After LC-MS TOF acquisition, DES levels were analyzed in the

extracted ion chromatogram.

Diethylstilbestrol (DES) inhibition of cholesterol binding

The sterol mixture was spiked with 79 μM DES, and the pull down for the targeted identification

of sterols was repeated as above using this mixture.

5
 Wei et al, SI

Circular Dichroism

Solutions of 6.36μM HIS-ERRα-LBD in 20mM Tris, 200mM NaCl, pH 8.0, 2% ethanol (v/v) were

incubated with 100μM small molecule for 5 minutes at 4°C, then circular dichroism was

measured on JASCO J-710 spectrophotometer at room temperature. Four measurements were

averaged.

Tryptophan Fluorescence Quenching

Tryptophan fluorescence quenching assay was carried out by incubation of varied concentration

of cholesterol with 1 µM His-ERRα-LBD in a total volume of 300 µL binding buffer (20 mM Tris

(pH 8.0), 150 mM NaCl and 5% ethanol (v/v)). Incubations were performed in 96-well black flat-

bottom plates and equilibrated overnight at 4 degrees. The samples were excited at 295 nm and

tryptophan fluorescence emission was collected at 332 nm.

Docking Simulations

Docking simulations were conducted with autodock vina using the ERRα LBD crystal structure

2JPL in the protein data bank (Trott and Olson, 2010). A cholesterol structure was generated in

ChemBioDraw. The simulation was centered at x coordinate -12, y coordinate -10, z coordinate

12. Simulation box size was x=22, y=22, z=22. The simulation was performed with a step size

(exhaustiveness) of 10.

Fluorescence Polarization Experiments

Fluorescence polarization experiments were performed by incubating 200 nM 25-NBD-

cholesterol or 6-NBD-cholesterol with 2.5-fold dilution of His-ERRα-LBD in 20 mM Tris (pH 8.0),

150 mM NaCl and 2.5% ethanol (v/v). Incubations were carried out in 384-well black flat-bottom

plates and equilibrated overnight at 4 degrees. Polarization was measured with λ(ex) = 485 nm

6
 Wei et al, SI

and λ(em) = 525 nm, and plotted using Prism software. Dissociation constants (Kd) were

determined by nonlinear regression analysis of dose-response curves.

Supplementary Reference

Bligh, E.G., and Dyer, W.J. (1959). A RAPID METHOD OF TOTAL LIPID EXTRACTION AND
PURIFICATION. Canadian Journal of Biochemistry and Physiology 37, 911-917.
Greschik, H., Althage, M., Flaig, R., Sato, Y., Chavant, V., Peluso-Iltis, C., Choulier, L., Cronet,
P., Rochel, N., Schϋle, R., et al. (2008). Communication between the ERRα Homodimer
Interface and the PGC-1α Binding Surface via the Helix 8-9 Loop. Journal of Biological
Chemistry 283, 20220-20230.
McDonald, J.G., Thompson, B.M., McCrum, E.C., and Russell, D.W. (2007). Extraction and
Analysis of Sterols in Biological Matrices by High Performance Liquid Chromatography
Electrospray Ionization Mass Spectrometry. Methods in Enzymology 432, 145-170.
Saghatelian, A., Trauger, S.A., Want, E.J., Hawkins, E.G., Siuzdak, G., and Cravatt, B.F.
(2004). Assignment of Endogenous Substrates to Enzymes by Global Metabolite Profiling†.
Biochemistry 43, 14332-14339.
Shan, H., Pang, J., Li, S., Chiang, T.B., Wilson, W.K., and Schroepfer Jr., G.J. (2003).
Chromatographic behavior of oxygenated derivatives of cholesterol. Steroids 68, 221-233.
Trott, O., and Olson, A.J. (2010). AutoDock Vina: improving the speed and accuracy of docking
with a new scoring function. efficient optimization and multithreading. Journal of
Computational Chemistry 31, 455-461.

7
 Wei et al, SI

A
Tissue Cholesterol Fold Enrichment
Brain 4.6*
Kidney 6.7*

Relative DES ion intensity

DES ion intensity

Figure S1. ERRα enriches cholesterol and diethylstilbestrol by affinity chromatography,

related to Figure 1. A. ERRα enriches cholesterol in both brain and kidney. Tissue sterols from
brain or kidney were isolated and then incubated with ERRα-LBD resin. After washing and
elution, ERRα-LBD was analyzed by targeted LC-MS to quantify ERRα-LBD bound sterols.
Cholesterol was the only sterol enriched by ERRα-LBD in both brain and kidney. * indicates p <
0.05 by student’s t-test. B. In a competitive binding assay, ERRα enriches diethylstilbestrol
(DES) from a lipid mixture spiked with DES. A mixture of the synthetic ERRα ligand
diethylstilbestrol (DES) with the total lipid extract was incubated with ERRα-LBD-bound resin or
a control (resin only). After washing and elution, an untargeted LC-MS analysis revealed the
enrichment of DES by ERRα-LBD. *** indicates p<0.005 by student’s t test.

8
 Wei et al, SI

Figure S2 Circular dichroism indicates that cholesterol, as well as synthetic ERRα

antagonists DES and XCT790, all induce conformational changes in ERRα-LBD, related to
Figure 1. Estradiol, a lipid known to not bind ERRα, does not induce a conformational change.
The upper image is a zoomed-in version of the same data in the bottom image.

9
 Wei et al, SI

Figure S3. Circular dichroism of the ERRα E235A F232A L228A mutant
demonstrates that the protein is folded and maintains an alpha helical secondary
structure, related to Figure 2. A characteristic profile for an alpha helix shows CD
minima at 208 and 222, whereas an unfolded protein will look like a random coil with
maxima (positive signal) at 212 and minima at around 195.

10
 Wei et al, SI

A 8
No HPCD
HPCD n.s.
0.6 n.s.
Fold (ERR PGC1/Vec)

n.s. n.s. n.s.

n.s. n.s. n.s.
n.s. n.s. n.s. **** n.s.
6
**** n.s.
Luc/-gal

0.4

Luc/ -gal
4

2 0.2

0
0 1 5 0.0
0 0 1 3 10 0 0 1 3 10 M
Lovastain (M)
Vec ERR +PGC1 Vec ERR +PGC1
n.s. - HPCD effect; n.s./n.s. - Lovastatin effect XCT790 Cholesterol

B No HPCD
HPCD
n.s.
n.s.
n.s.
20
n.s. n.s. 0.8 n.s.
Fold (ERRPGC1/Vec)

n.s. n.s. n.s. n.s. n.s. n.s. **** n.s.

****
15
0.6
Luc/ -gal

Luc/-gal
10
0.4

5
0.2

0
0 1 5 0.0
0 0 1 3 10 0 0 1 3 10 M
Lovastain (M)
Vec ERR+PGC1 Vec ERR+PGC1
n.s. - HPCD effect; n.s./n.s. - Lovastatin effect XCT790 Cholesterol

C 40
No HPCD
HPCD n.s. n.s.
0.25 n.s. n.s.
n.s. n.s.
**** n.s. **** n.s.
Fold (Nur77/Vec)

30 n.s. n.s. n.s. n.s. 0.20

n.s.
Luc/-gal

Luc/-gal

0.15
20
0.10
10
0.05
0
0 1 5 0.00
0 0 1 3 10 0 0 1 3 10 M
Lovastain (M)
Vec Nur77 Vec Nur77
n.s. - HPCD effect; n.s./n.s. - Lovastatin effect XCT790 Cholesterol

Figure S4. Cholesterol does not affect the transcriptional activity of other orphan nuclear
receptors, related to Figure 3. Selected orphan nuclear receptors were examined by transient
transfection and reporter assays in a similar fashion as in Figure 3A-B. A. ERRβ. B. ERRγ. C.
Nur77.

11
 Wei et al, SI

A 0.05
IDH3a Veh
0.05
VLCAD
0.4
TRAP
0.03
Ctsk
0.04 XCT790 0.04 ***

Relative mRNA
0.3
Relative mRNA

Relative mRNA
**

Relative mRNA
** **
0.02
0.03 0.03
n.s. 0.2
n.s.
0.02 n.s. 0.02 n.s.
0.01
0.1
0.01 0.01

0.00 0.00 0.0 0.00

WT ERRaKO WT ERRaKO WT ERRaKO WT ERRaKO

B 1.5
ERR in C2C12
1.5
PGC1 in C2C12
C 1.5
ERR in osteoclast
1.5
PGC1 in Osteoclast

n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

Fold in relative mRNA

Fold in mRNA
1.0 1.0 1.0 1.0

0.5 0.5 0.5 0.5

0.0 0.0 0.0 0.0

Veh Simva Simva+Chol Veh Simva Simva+Chol Veh Lova Lova+Chol Veh Lova Lova+Chol

WT-V Veh-V
D 10 IL-1
*
WT-LPS
ERRaKO-V
ERRaKO-LPS
E 10 IL-1
Veh-LPS
Cholesterol-V
Cholesterol-LPS
**
8 8
Relative mRNA

n.s. n.s. n.s. n.s.

n.s. n.s. n.s.
n.s. 6
6

4 4
** ****
2 2

0 0
Veh Chol Lova Lova/Chol Zole Zole/Chol Veh XCT790
25 MMP9 20 MMP9
**
***
20 15 n.s.
Relative mRNA

n.s. n.s. n.s.

15 n.s. n.s. n.s. n.s.
10
10 ****
*** 5
5

0 0
Veh Chol Lova Lova/Chol Zole Zole/Chol Veh XCT790

F 1.5 Dhcr24 Veh 1.5 Hmgcr Veh 5 Abcg1 Veh 3 Abca1 Veh
Fold (mRNA in macrophage)

Chol Chol Chol Chol

**
4 **
** **
1.0 1.0 2
3

2
0.5 0.5 *** ** 1
*** ***
1

0.0 0.0 0 0
WT ERRaKO WT ERRaKO WT ERRaKO WT ERRaKO

Figure S5. Additional Cellular Analysis, related to Figure 4 and 5. A. XCT790 effects on
ERRα target genes (IDH3a and VLCAD) and osteoclast differentiation markers (TRAP and Ctsk)
were abolished in ERRαKO osteoclast differentiation cultures. B. Expression of ERRα and
PGC1α in C2C12 cultures were unaltered by simvastatin or cholesterol. Cells were treated as in
Figure 4G. C. Expression of ERRα and PGC1β in osteoclast cultures were unaltered by
lovastatin or cholesterol. Cells were treated as in Figure 5D. D. Regulation of other inflammatory
markers by cholesterol, statin and bisphosphonate was abolished in ERRαKO macrophages.
Macrophages were cultured as in Figure 5E. E. Cholesterol suppression of other inflammatory
markers was abolished by XCT790. Macrophages were cultured as in Figure 5F. F. Cholesterol
regulation of SREBP and LXR target genes were unaltered in ERRαKO macrophages. Cells
were treated with 10μM cholesterol.

12
 Wei et al, SI

n.s.
*
n.s.
5 10 6 * 2.0 10 4 1.0 10 4 3 10 4
*
4 10 6 * ** 8.0 10 3 2 10 4
1.5 10 4
3 10 6 6.0 10 3 2 10 4
1.0 10 4
2 10 6 4.0 10 3 1 10 4
5.0 10 3
1 10 6 2.0 10 3 5 10 3

0 0 0 0
Cholesterol 7 -OH-Chol 7-keto-Chol 4 -OH-Chol

4 10 4 4 10 3 8 10 5 4 10 5

3 10 4 3 10 3 6 10 5 3 10 5
Ion Intensity

2 10 4 2 10 3 4 10 5 2 10 5

1 10 4 1 10 3 2 10 5 1 10 5

0 0 0 0
24, 25-epoxy-Chol 24-S/25/24-R-OH-Chol 5,6 -epoxy-Chol 5,6 -epoxy-Chol

2.0 10 4 2.0 10 4 8 10 3

1.5 10 4 1.5 10 4 6 10 3

1.0 10 4 1.0 10 4 4 10 3

5.0 10 3 5.0 10 3 2 10 3

0 0 0
Cholestenone Desmosterol 27/22-S-OH-Chol

Figure S6. LC-MS analysis of sterols in osteoclast differentiation cultures, related to

Figure 5. RAW264.7 mouse macrophage precursors were differentiated into osteoclasts with
100ng/ml RANKL for 2 days, and treated with cholesterol, lovastatin and/or zoledronate as in
Figure 5A-B. Sterols were quantified by targeted LC-MS (n=4).

13
 Wei et al, SI

A Veh
100

Serum Cholesterol (mg/dL)

Zole

80
* *
60

40

20

0
WT ERRaKO

B 2.5 Aco2 2.5 IDH3a

Fold (mRNA in osteoclast)

Chow Chow
*** HCD * HCD
2.0 2.0

1.5 1.5

1.0 1.0

0.5 n.s. 0.5 n.s.

0.0 0.0
WT ERRaKO WT ERRaKO

C
2.0 MCAD 2.0 Aco
Chow Chow
Fold (mRNA in muscle)

** HCD * HCD
1.5 1.5

1.0 1.0
n.s.
n.s.
0.5 0.5

0.0 0.0
WT ERRaKO WT ERRaKO

D
5 Fbxo32 1.5 Myh2 Veh
Fold (mRNA in muscle)

Veh + Dex
4 Dex **
1.0
3 *** ++
++
**
2 ++
0.5 **
1

0 0.0
WT ERRaKO WT ERRaKO

Figure S7. Additional In Vivo Analysis, related to Figure 4 and 6. A. Serum cholesterol
levels. Mice were treated as in Figure 6A. B-C. Expression of ERRα target genes in osteoclast
(B) and muscle (C). Mice were treated as in Figure 6B. D. Glucocorticoid-induced myopathy
was intact in ERRα-KO mice. ERRα-KO mice and WT controls (4 month old, male, n=6) were
treated with 10mg/kg/day dexamethasone (Dex) or vehicle (Veh) control via daily intraperitoneal
injection for 10 days.

14
 Wei et al, SI

Sterol Transition Detected in Sterol Mixture Fold change ERRα/Resin

Saturated Tetraols 454.4 to 367.3 no N/A
Unsaturated Tetraol 452.4 to 365.3 no N/A
Lanosterol 444.4 to 409.4 yes N/A
Saturated Triols A 438.4 to 385.3 yes 0.9
Saturated Triols B 438.4 to 367.3 yes N/A
Saturated Triols C 438.4 to 420.4 no N/A
Unsaturated Triols A 436.4 to 418.4 no N/A
Unsaturated Triols B 436.4 to 383.3 yes N/A
Unsaturated Triols C 436.4 to 365.3 no N/A
24-dihydrolanosterol 429.4 to 411.4 no N/A
Hydroxy/Epoxy Sterols A 420.4 to 367.3 yes 1.5
Hydroxy/Epoxy Sterols B 420.4 to 385.3 yes 0.8
24-25 Epoxy Sterols 418.4 to 383.3 yes N/A
cholestanol 406.4 to 371.4 yes 1.2
Cholesterol 404.4 to 369.4 yes 4.6
Desmosterol 402.4 to 367.3 yes N/A
7-ketochol 401.3 to 383.3 no N/A
unsaturated mixture 385.3 to 367.3 no N/A
7α Hydroxy Cholesterol 385.3 to 367.3 yes N/A

Table S1. Cholesterol is the only sterol that was enriched by ERRα LBD, related to Figure
1. A large range of sterols in the tissue sterol mixture were examined by MRM mass
spectrometry before and after pull-down. Several sterol derivatives were identified in the sterol
mixture prior to pull-down whereas they could not be identified in the ERRα or resin only
samples (N/A). An additional set of sterols was examined, but was not found to be present at
measurable concentrations in the sterol extract.

15