LC-MS Determination of Bile Acid-Sulfates As Biomarkers for Liver Function

1
LC-MS Determination of Bile Acid-Sulfates As
Biomarkers for Liver Function

• Yazen Alnouti, Ph.D
• Assistant Professor
• Department of Pharmaceutical Sciences
• University of Nebraska Medical Center

2
BA Enterohepatic Recirculation
95
Under normal conditions, BAs are contained within
the enteroheaptic system, spill over into blood
is minimum, and urinary excretion is negligible
3
Backbone Structure
R1 R2 R3 R4
Tri-OH-Bile Acids Tri-OH-Bile Acids Tri-OH-Bile Acids Tri-OH-Bile Acids
a-Muricholic acid ß-OH a-OH H OH
ß-Muricholic acid ß-OH ß-OH H OH
?-Muricholic acid a-OH ß-OH H OH
Cholic acid H a-OH a-OH OH
Di-OH-Bile Acids Di-OH-Bile Acids Di-OH-Bile Acids Di-OH-Bile Acids Di-OH-Bile Acids
Ursodeoxycholic acid H ß-OH a-OH OH
Chenodeoxycholic acid H a-OH H OH
Deoxycholic acid H H H OH
Mono-OH-Bile Acids Mono-OH-Bile Acids Mono-OH-Bile Acids Mono-OH-Bile Acids Mono-OH-Bile Acids
Lithocholic acid H H H H
4
Increasing Interest in Bile Acids
5
Eras of Interest in BAs

1. Lipid Absorption/Cholesterol Elimination
2. Cytotoxicity
3. Genotoxicity
4. Cholestasis/ Liver Toxicity
5. Colon Cancer
6. Treatment of hepatobiliary diseases
7. Signaling Molecules (Hormones) nuclear receptors
   ligands

• BA Homeostasis (Cholestasis)
• Glucose Homeostasis (Diabetes)
• Energy Homeostasis (Metabolic Syndrome)

6
Missing with BA Homeostasis Can be the Kiss of
Death

• Bile/Liver 64 mM/0.45 mM 150 fold
• Liver/Plasma 0.45 mM /1.2 mM 400 fold
• Bile/Plasm 64 mM /1.2 mM 60,000 fold

7
BA Homeostasis
BA homeostasis is tightly controlled by nuclear
receptor-mediated mechanisms which up- and/or
down-regulate target genes involved in BA i)
Synthesis ii) Transport iii) Metabolism
8
BA Homeostasis/Sulfation

• Sulfate-conjugates carry a permanent negative
  charge (pKa of sulfate moiety lt 1)
• Efficient pathway of BA detoxification
• i. Decrease intestinal absorption
• ii. Increase fecal excretion
• iii. Increase urinary excretion
• iv. Decrease toxicity

9
BA Sulfation in Humans

• Under normal conditions, total BAs excreted in
  urine is lt1 mMole/day (40-70 sulfated), total
  BAs in plasma lt 3 mM (lt10 sulfated), up to 4 of
  BAs in bile are sulfated
• LCA (mono-hydroxy BA) is almost exclusively
  present in the sulfated form, whereas CA
  (Tri-hydroxy BA) is mostly present in the
  unsulfated form.
• In hepatobiliary/cholestatic diseases, due to
  the impairment of biliary excretion, BAs in
  blood, tissues, and urine increase
• - Amounts of BA-sulfates also increase In bile,
  urine (gt100x), plasma
• - Concluded that BA sulfation is induced during
  hepatobiliary diseases to protect against BA
  accumulation and toxicity
• - of BA-sulfates/BAs decrease/increase ?!
  (missing)

10
Marked Variation on Data on BA-Sulfates in Health
Disease
Alnouti Y. Toxicol Sci. 2009 Jan 8. Epub ahead
of print
11
Analysis of BA-Sulfates

• The variation in Data on BAs and BA-sulfates is
  largely due to the analytical techniques used to
  quantify them
• BA-sulfates are desulfated during sample
  preparation, then unconjugated BAs are quantified
  by
• i. 3-a-OH-steroid dehydrogenase (3aHSD)
• ii. GC/GC-MS
• iii. LC-UV/Fluorescence/(3aHSD)
• iv. LC/MS-(FAB, ESI)
• - These methods are not direct and they lack
  specificity, sensitivity, validation, and/or
  dynamic range

12
UPLC-MS/MS
13
Representative Chromatogram of Bile Acid Mixture
G-CA
G-CA
2
1. G-UDCA 2. G-CDCA 3. G-DCA
3
1
G-LCA
2
1. T-MCA 2. T-CA
1
Intensity
1
3
1. T-UDCA 2. T-CDCA 3. T-DCA
2
T-LCA
2
1. a-MCA 2. b-MCA 3. CA
3
1
1. T-UDCA 2. T-CDCA 3. T-DCA
2
3
1
LCA
2H4-G-CDCA (IS)
2H4-CDCA (-IS)
Time (Min)
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Bile BA Concentrations
15
Current Future BA-related Research in my Lab

• LC-MS/MS method for the quantification of
  BA-sulfates
• . MS conditions direct vs. indirect (scanning)
• . Sample Preparation SPE, PP, LLE..etc
• . Chromatography column, mobile phase,
  wash..etc
• . Validation accuracy, precision,
  stability..etc
• SULT2A1 Cell Line in vitro kinetics of
  sulfation of BAs and others!
• Synthesis/Biosynthesis of BA-Sulfate standards
• - Hepatotoxicity of endothelin receptor-2
  antagonists

16
Significance

• Need-Driven
• There is a critical need for direct, sensitive,
  valid, specific, simple, and fast bioanalytical
  methodology for the quantification of BAs and
  BA-sulfates in biological matrices
• ii. Hypothesis-Driven
• BA-sulfates can be used as a biomarker for liver
  function in hepatobiliary diseases

17
Specific Aims
i. Develop and Validate a rapid, specific, and
sensitive LC-MS/MS method to quantify individual
BA and BA-sulfates ii. Bile, urine, plasma
analysis from patients with hepatobiliary
diseases (liver transplant) iii. Correlation
Analysis of individual BA and BA-sulfates
18
Novelty
i. No direct and valid methodology to quantify
specific BA sulfates in biological fluids and
tissues ii. No selective biomarker available to
diagnose and predict the outcomes of liver
disease treatment/transplant
19
Predicted Outcomes/ Fit to COBRE

• Development and validation of a novel rapid
  bioanalytical methodology for the analysis of
  BA-sulfates in biological matrices, which will be
  available for scientists in BA research
• The application of this methodology is expected
  to lead to the discovery of a novel biomarker for
  liver function
• The biomarker which can be used is the diagnosis
  and treatment of several hepatobiliary diseases
  and/ or liver transplant

20
Bile acid toxicity in man hepatobiliary

• Accumulation of intermediates in bile acid
  biosynthesis in hepatocytes because of inborn
  enzyme deficiencies
• Elevated concentration in hepatocytes because of
  defective canalicular transport (BSEP deficiency,
  cholestasis of pregnancy)
• Elevated concentrations in cholangiocytes because
  of bile duct obstruction (PBC, PSC)

21
Foreläsningar i Djurkemien, 1808 The function of
bile in the body is to digest chyme in the
duodenum. The bile substance remains in solution
to be resorbed and used again.
22
Eras of Interest in BAs

1. Lipid Absorption/Cholesterol Elimination
2. Cytotoxicity/Genotoxicity
3. Cholestasis/Liver Toxicity
4. Colon Cancer
5. Treatment of hepatobiliary diseases
6. Signaling Molecules (Hormones) nuclear receptors
   ligands

• BA Homeostasis (Cholestasis)
• Glucose Homeostasis (Diabetes)
• Energy Homeostasis (Metabolic Syndrome)

23
Bile Acids Are Nuclear Receptor Ligands
24
Bile Acids Are Very Different

• Lipophylicity (membrane destruction) LCAgtgtgtCA
• DCA causes cholestasis, whereas CA causes
  hypercholeresis
• Cytotoxicity/genotoxicity LCAgtgtgtCA
• Hepatobiliary Toxicity UDCA treats gallstone,
  whereas LCA is very cytotoxic
• FXR ligands CDCAgtgtgtLCA
• PXR, VDR ligands LCAgtgtgtCDCA

25
Backbone Structure
R1 R2 R3 R4
Tri-OH-Bile Acids Tri-OH-Bile Acids Tri-OH-Bile Acids Tri-OH-Bile Acids
a-Muricholic acid ß-OH a-OH H OH
ß-Muricholic acid ß-OH ß-OH H OH
?-Muricholic acid a-OH ß-OH H OH
Cholic acid H a-OH a-OH OH
Di-OH-Bile Acids Di-OH-Bile Acids Di-OH-Bile Acids Di-OH-Bile Acids Di-OH-Bile Acids
Ursodeoxycholic acid H ß-OH a-OH OH
Chenodeoxycholic acid H a-OH H OH
Deoxycholic acid H H H OH
Mono-OH-Bile Acids Mono-OH-Bile Acids Mono-OH-Bile Acids Mono-OH-Bile Acids Mono-OH-Bile Acids
Lithocholic acid H H H H
26
BA Synthesis
Classic/Neutral
CYP8B1
7-OH- Cholesterol
Intestine
CA
CYP7A1
DCA
glycine/taurine Conjugates
Cholesterol
Intestine
CYP27A1
CYP46A1, 25
LCA
CYP7B1
7, 27-DiOH-Cholesterol
CDCA
Oxysterol
CYP39A1
Alternative/Acidic
UDCA (3a, 7b-di-OH) Bears
MCA (3a, 6a/b,7a/b-tri-OH) Rodents
27
BA Metabolism/Homeostasis
Hydroxylation
Conjugation
Transporters
Synthesis
28
NTCP
OATP?
BA
OA
OCT
OC
MDR2/3
MDR1
Cl-
AE
PS
HCO3-
Bile flow
FIC1
PC
PL
BA
ASBT
H2O
Cl-
Ch
OA
AQP
BSEP
BA
ABCG5/8
Cholangiocyte
MRP2
Hepatocyte
MRP4
Figure from Alan F. Hofmann, University of
California Sann Diego
29
Bile acid toxicity in man hepatobiliary

• Accumulation of intermediates in bile acid
  biosynthesis in hepatocytes because of inborn
  enzyme deficiencies
• Elevated concentration in hepatocytes because of
  defective canalicular transport (BSEP deficiency,
  cholestasis of pregnancy)
• Elevated concentrations in cholangiocytes because
  of bile duct obstruction (PBC, PSC)

30
Hepatobiliary Bile Acid Toxicity in Experimental
Animals

• Increased concentration of lithocholic acid (LCA)
  because of feeding CDCA (or UDCA) and defective
  detoxification of LCA (mouse, rhesus monkey and
  baboon)
• Elevated concentrations of simple BA micelles in
  bile causing cholangiocyte destruction because of
  absent phospholipid in bile (MDR2 deficiency)
  (mice)
• Increased concentration of deoxycholic acid and
  its 3-oxo derivative because of taurocholate
  feeding cause lethality in guinea pig

Figure from Alan F. Hofmann, University of
California Sann Diego
31
Drugs can inhibit bile acid transporters a kiss
of death

• Assay
• Polarized monolayers in which NTCP and BSEP have
  been transfected
• Testing of drugs for inhibition of uptake via
  NTCP or canalicular secretion via BSEP
• Fluorescent bile acids for high throughput
  screening
• (laboratory of Yuichi Sugiyama, University of
  Tokyo)

32
How BAs Are Quantified?

1. 3-a-OH-steroid dehydrogenase (3aHSD)
2. GC/GC-MS
3. LC-UV/Fluorescence/(3aHSD)
4. LC/MS-(FAB, ESI)

33
Enzymatic Determination of 3a-hydroxy Bile Acids
HPLC of human biliary bile acids
Origins Talalay, Iwata and Yamasaki, Javitt,
Palmer
34
There is a critical need for valid, accurate, and
simple analytical methodology for the
quantification of BAS and their metabolites in
biological matrices
35
LC-MS/MS
36
LC-MS
37
Method Development

• MS Conditions
• Chromatography Conditions
• Sample Preparation
• Method Validation

38
MS Conditions
Compound Dependent Parameters

• Capillary Voltage 3 kV
• Cone Voltage Compound dependent
• Extractor Voltage 4 V
• RF lens 0 V
• Source Temperature 120 C
• Desolvation Temperature 375 C
• Desolvation Gas Flow 500 L/hr
• Cone Gas Flow 65 L/hr
• Collision Energy Compound dependent
• Polarity Compound dependent

  Cone Voltage (V) Collision Energy (eV) Polarity
G-CA 25 20
G-UDCA 20 18
G-CDCA 20 18
G-DCA 20 18
G-LCA 17 15
T-MCA 90 70 /-
T-CA 90 70 /-
T-UDCA 78 65 /-
T-CDCA 78 65 /-
T-DCA 78 65 /-
T-LCA 71 63 /-
a-MCA 65 15 -
b-MCA 65 15 -
CA 65 15 -
UDCA 60 17 -
CDCA 60 17 -
DCA 60 17 -
LCA 57 20 -
39
MS/MS Transitions
_
_

40
Chromatography Conditions
41
Final LC Conditions
The mobile phase consisted of 5 acetonitrile
(ACN) in methanol (MeOH) (mobile phase A) and
7.5 mM ammonium acetate adjusted to pH 4 using
acetic acid (mobile phase B) at a total flow rate
of 0.3 ml/min.
42
Representative Chromatogram of Bile Acid Mixture
G-CA
G-CA
2
1. G-UDCA 2. G-CDCA 3. G-DCA
3
1
G-LCA
2
1. T-MCA 2. T-CA
1
Intensity
1
3
1. T-UDCA 2. T-CDCA 3. T-DCA
2
T-LCA
2
1. a-MCA 2. b-MCA 3. CA
3
1
1. T-UDCA 2. T-CDCA 3. T-DCA
2
3
1
LCA
2H4-G-CDCA (IS)
2H4-CDCA (-IS)
Time (Min)
43
Representative Bile Chromatogram
Intensity
Time (Min)
44
Quantification of Endogenous Analytes is Hard

• There is no blank matrix to construct
• a calibration curve?!

• Labeled standards of each analyte
• Method of standard addition
• Matrix stripping

45
Sample Preparation

• Liver Alkaline-ACN protein precipitation
• Plasma ACN protein precipitation
• Urine Solid Phase Extraction (SPE)
• Bile Solid Phase Extraction (SPE)

46
Extraction Recoveries(extraction efficiency
matrix effect/suppression)
47
Method Validation

• Limit of detection (LOD) quantification (LOQ)
  
• Specificity carry over
• Stability (bench top, freeze/thaw, autosampler)
• Matrix effect
• Dynamic range
• Goodness of fit
• Accuracy and precision
• Ruggedness

48
BA Calibration Curves
T-DCA
G-CA
T-MCA
G-UDCA
MDCA
G-CDCA
CA
G-DCA
UDCA
T-CA
T-UDCA
CDCA
T-CDCA
UDCA
49
Accuracy Precision
50
Applications

• Control Mice BA profiling in mouse tissues and
  fluids
• Male vs. female mice
• Bosentan toxicity (Jaeschke)
• FXR null mice (Guo)
• BAs toxicity/dose response (Klaassen)

51
Liver BA Concentrations
52
Bile BA Concentrations
53
Biliary BA Excretion
54
Cumulative Biliary BA Excretion
55
Plasma BA Concentrations
56
of Individual BAs
57
How does our data compare to literature?

• Plasma 600 ng/ml (204-7650)
• Liver 225 mg/g (20-618)
• Bile 20 mg/ml (16-102 )

58
This why Missing with BA transportes can ne the
Kiss of Death

• Bile/Liver 64 mM/0.45 mM 150 fold
• Liver/Plasma 0.45 mM /1.2 mM 400 fold
• Bile/Plasm 64 mM /1.2 mM 60000 fold

59
No BAs in Urine
Bile
Liver
Plasma
Urine
60
Unless Urine is Contaminated with Feces
61
Polar BA Species(No standards available)
T-3OH-G
2OH-G
T-1OH-S
T-2OH-G
T-1OH-G
1OH-G
T-3OH-S
T-4OH
3OH-S
3OH-G
T-2OH-S
2OH-S
62
Male vs. Female BAs
Liver
Bile
63
Biliary BAs in Young and Old FXR KO Mice

• WT mice, no age difference
• Except for G-cholic, alpha-muri, and T-urso that
  are higher in older mouse bile, and T-cheno that
  is higher in younger mouse bile.
• Old FXR KO gt young FXR KO and old and young WT
  mice
• G-cholic
• T-muri
• Cholic
• T-cholic
• Beta-muri
• Young FXR KO gt WT mice
• G-cholic
• T-cholic
• Maybe T-urso
• Young FXR KO gt old FXR KO gt WT mice
• T-deoxy
• Unchanged in FXR KO mice

64
Bosentan

• Endothelin receptor antagonist (pulmonary
  hypertension)
• Hepatotoxicity in Humans (ALT levels)
• Differential Specie-toxicity
• Increase in serum BAs in humans
• Unknown mechanism (Ntcp, Mrp2, Bsep)?

65
Bosentan effect on biliary BAs
66
Bosentan/Plasma BAs
No Effect of Bosentan on Plasma BAs
67
Conclusion

• We have a sensitive and valid LC-MS method to
  quantify 6 BAs and their glycine and taurine
  metabolites in biological fluids and tissues.
• The wide variation in results of BA analysis is
  due to the analytical methodology and sample
  collection.
• Biliary BAs 150x gt liver 400x gt plasma ?gt urine.
• Muricolic and colic acids in the taurine
  conjugate form represents gt 95 of total BAs.
• Rodents produce minimal amounts of glycine
  conjugates.
• Polar BA metabolites should be considered and
  may be more significant under abnormal
  conditions.
• This methodology is reliable for a wide variety
  of applications.