WO2019185738A1

WO2019185738A1 - Method for the diagnosis of biliary tract diseases

Info

Publication number: WO2019185738A1
Application number: PCT/EP2019/057757
Authority: WO
Inventors: Bryn FLINDERS; Ronald Martinus Alexander Heeren; Stephanus Willibrordus Maria Olde Damink; Franciscus Gerardus Schaap; Robert Jan VREEKEN
Original assignee: Universiteit Maastricht; Academisch Ziekenhuis Maastricht
Priority date: 2018-03-27
Filing date: 2019-03-27
Publication date: 2019-10-03

Abstract

The present invention relates to an in vitro method for diagnosing a biliary tract disease in a subject by determining the concentration of one or more biomarkers in a biospecimen from the subject and comparing it to a reference biospecimen. The method is particularly suitable for diagnosing primary sclerosing cholangitis (PSC) and cholangiocarcinoma. The preferred detection method for the biomarkers is MALDI- MSI.

Description

METHOD FOR THE DIAGNOSIS OF BILIARY TRACT DISEASES

The present invention relates to a method for diagnosing a biliary tract disease. In particular the invention relates to determining certain biomarkers in a biospecimen from a subject using e.g. a MALDI technique in order to determine the risk of the subject of having or developing a biliary tract disease.

The biliary tract refers to the organs and ductular network that make and store bile and release it into the small intestine. The biliary tract includes the bile ducts inside and outside the liver and the gallbladder.

Examples of diseases of the biliary tract are inflammation of gall bladder and bile duct and malignant or benign tumours in any of the organs and biliary ducts.

There is a need for improved diagnostic methods of the above mentioned diseases. Preferably these diagnostic methods are non-invasive, sensitive and specific.

The present invention thus provides an in vitro method for diagnosing a biliary tract disease in a subject comprising the steps of:

a) determining the concentration of one or more biomarkers for a biliary tract disease in a biospecimen from the subject;

b) comparing the concentration of the one or more biomarkers in the biospecimen from the subject with the concentration of the one or more biomarkers in a reference biospecimen;

c) diagnosing the subject with the biliary tract disease if the concentration of the one or more biomarkers in the biospecimen from the subject is different from the concentration of the one or more biomarkers in the reference biospecimen, wherein the one or more biomarkers are selected from the groups consisting of sulfatides and 5-cyprinol sulfate.

The diseases that can be diagnosed with the method of the invention are biliary tract diseases, more in particular primary biliary cholangitis, primary sclerosing cholangitis (PSC), intrahepatic, perihilar and distal cholangiocarcinoma, cholecystitis, choledocholithiasis, and gallbladder cancer. The method is particularly suitable for the diagnosis of cholangiocarcinoma and diagnosis of primary sclerosing cholangitis.

With diagnosis is meant that it is determined that the subject suffers from a disease or not, or it is determined that the subject is at a certain stage of the disease, i.e. the severity of the disease is determined. The biospecimen to be used for the method of diagnosis of the invention can be a tissue sample or a biofluid. In one embodiment, the biospecimen is a sample of biliary tract tissue, preferably liver, gall bladder or bile duct tissue. More preferably, the biospecimen is a sample of liver tissue. In another embodiment, the biospecimen is a biofluid, in particular blood plasma, blood serum, bile, urine, luminal contents of the intestines or faeces.

In case a tissue sample is used as the biospecimen, it can be prepared according to methods known in the art. In the case of liver tissue, the tissue can be sectioned using known techniques to produce sections in the order of micrometres which can be mounted on glass slides.

In the method of the invention, the biospecimen of the subject is compared to a reference biospecimen. As the reference biospecimen the same type of tissue or biofluid is taken as that of the subject where a diagnosis needs to be made, but the reference biospecimen is taken from a healthy subject, i.e. a subject known not to have the disease to be diagnosed. Alternatively, if the reference biospecimen is a tissue sample, it can be taken from a non-diseased biliary tract tissue of the subject.

In the above, the subject is preferably a mammal, most preferably a human.

The invention also relates to a kit for the diagnosis of a biliary tract disease, wherein the kit contains:

• means for collecting a biospecimen;

• means for measuring 5-cyprinol sulfate or sulfatides in the biospecimen;

• optionally, instructions for using the kit;

• optionally, a reference sample.

According to a first aspect, the present invention thus provides an in vitro method for diagnosing a biliary tract disease in a subject comprising the steps of : a) determining the concentration of bile salts and bile alcohols in a biospecimen from the subject;

b) comparing the concentration of bile salts and bile alcohols in the biospecimen from the subject with the concentration of bile salts and bile alcohols in a reference biospecimen;

c) diagnosing the subject with the biliary tract disease if the concentration of bile salts and bile alcohols in the biospecimen from the subject is different from the concentration of bile salts and bile alcohols in the reference biospecimen, wherein the bile alcohol is 5-cyprinol sulfate. Bile salts (also referred to as bile acids) and bile alcohols, collectively called the cholanoids, are end-metabolites of cholesterol that show striking cross species diversity in chemical structure, ranging from C₂7-bile alcohols in phylogenetically basal vertebrates (e.g., jawless fish, cartilaginous fish) to C₂₄-bile salts in most birds, reptiles, and mammals.

Bile alcohols are the main cholanoids of all jawless and cartilaginous fish analyzed to date and are also found in some teleost fish and amphibians, as well as a small number of species of birds, reptiles, and mammals. Bile alcohols are typically secreted from the liver as sulfate conjugates. The stereochemistry of the juncture between the A and B rings of cholanoids is variable and influences the overall shape of the bile salt/alcohol, with 53-cholanoids having a“bent” orientation, and 5a(‘allo’)-cholanoids having a flat (planar) structure.

f% ,-5(t-Bilc acid ί^', ,-50-Bile acid

(Cholic acid) (Alloeliolic acid l

The preferred bile alcohol, 5-cyprinol sulfate has the following structure:

5-cyprinol sulfate includes the a-form, the b-form and mixtures thereof.

The present invention thus also relates to 5-cyprinol sulfate for use in the diagnosis of a biliary tract disease. Preferably, the 5-cyprinol sulfate has a purity of at least 95%, more preferably at least 99%, most preferably at least 99.5% or even 100%. Thus, if cyprinol sulfate is used as a standard (reference) in carrying out the diagnosis, a relatively pure form is used, not containing any further components, even when the cyprinol sulfate is originally obtained from a natural source such as fish.

As described above, the method of the invention comprises determining the concentration of 5-cyprinol sulfate in a biospecimen from the subject and comparing to a reference biospecimen. The difference in concentration of 5- cyprinol sulfate between the subject biospecimen and the reference can be an increase in concentration or a decrease in concentration.

Several methods are available to detect 5-cyprinol sulfate in the biospecimen. If the biospecimen is a tissue sample, in particular from the liver, gall bladder or bile duct, 5-cyprinol sulfate is determined by a MALDI-MSI (Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) method. MALDI-MSI allows to monitor the spatial distribution of bile salts/alcohols and lipids in tissue.

In case of the use of MALDI-MSI, a tissue is sectioned to a tissue preparation with a thickness of several pm, e.g. 1 -20 pm. This tissue section is then mounted onto a glass slide. Subsequently, after desiccation, the tissue section on the glass slide is coated with an appropriate matrix material., e.g. 2,5-dihydrobenzoic acid (DHB), e.g. by spraying a solution of the matrix material in a suitable solvent, e.g. methanol, onto the tissue section on the glass slide.

The tissue section coated with matrix material can then be subjected to MALDI-MSI analysis.

If the biospecimen is a biofluid, cyprinol sulfate concentrations can be determined by known chromatography methods. Examples are U(H)PLC-MS (Ultra (High) Performance Liquid Chromatography) and U(H)PLC-MS/MS (Ultra (High) Performance Liquid Chromatography tandem Mass Spectometry).

Preferably the method of diagnosis of the invention is an in vitro method.

As will be shown hereafter, in one embodiment, the present invention is particularly suitable for diagnosing primary sclerosing cholangitis (PSC) in a subject comprising the steps of:

a) determining by means of a MALDI method the concentration of 5-cyprinol sulfate in a biospecimen from the subject, wherein the biospecimen is a liver tissue;

b) comparing the concentration of 5-cyprinol sulfate in the biospecimen from the subject with the concentration of 5-cyprinol sulfate in a reference biospecimen;

wherein an increase of the concentration of 5-cyprinol sulfate in the biospecimen from the subject relative to the reference biospecimen indicates that the subject has an altered risk of having or developing PSC.

According to a second aspect, the present invention provides an in vitro method for diagnosing a biliary tract disease in a subject comprising the steps of : a) determining the concentration of sulfatides in a biospecimen from the subject; b) comparing the concentration of sulfatides in the biospecimen from the subject with the concentration of sulfatides in a reference biospecimen;

c) diagnosing the subject with the biliary tract disease if the concentration of sulfatides in the biospecimen from the subject is different from the concentration of sulfatides in the reference biospecimen.

As described above, in the method of the invention, sulfatides are detected in a biospecimen in order to diagnose biliary tract disease. The biological role and structure of sulfatides is for instance described in T. Takahashi et al, J. of Lipid Research, Vol. 53, 2012, p. 1437-1448. Sulfatides are sometimes also referred to as glycosphingolipid sulfates.

As described in T. Takahashi et al., the sulfatides exhibit various structures, including different lengths of the acyl chain and ceramide moiety, which can be hydroxylated, as well as other sphingolipids. In detail, the major sulfatides are composed of ceramides possessing 4-sphingenine (d18:1 ) with either C22-, C23- or C24- hydroxylated fatty acids, viz. (C22:0 OH), (C23:0 OH), (C24:0 OH), or (C24:1 OH), or with C24 non- hydroxylated fatty acids (C24:0) and (C24:1 ). The minor sulfatides are composed of ceramides possessing 4-sphingenine (d18:1 ) with either C16-, C18-, C20-, C21 -, C22- or C23- hydroxylated fatty acids, viz. (C16:0 OH), (C18:0 OH), (C20:0 OH), (C21 :0 OH), (C22:0 OH), (C23:0 OH), or C16:0-, C18:0-, 020:0-, C21 :0-, C22:1 -, 022:0-, 023:0-, 026:1 - or 026:0 non-hydroxylated fatty acids, viz. (C16:0, C18:0, C20:0, C21 :0, C22:1 , C22:0, C23:0, C26:1 , and 026:0 fatty acids and phytosphingosine (t18:0) with C20- or C24- hydroxylated fatty acids, viz. (020:0 OH) or (024:0 OH)..

According to the invention, in particular the following sulfatides are detected (as deprotonated molecules, viz. [M-H]-ions):

[ST-OH (18:1 24:0)],

[ST-OH (18:1 _ 16:0)],

[ST-OH (18:1 _ 24:1 )],

[ST-OH (18:1 _22:0)],

[ST(42:0)],

[ST(41 :1 )],

[ST (42 : 1 ) ;

[ST (18:1 _ 16:0)],

[ST (18:1_24:0)] and

[ST (40:0)].

More preferably, the sulfatides (detected as deprotonated molecule) to be determined are selected from

[ST-OH (18:1 _ 24:0)];

[ST-OH (18:1 _16:0)];

[ST-OH (18:1 _ 24:1 )]; and

[ST-OH (18:1 22:0)].

Most preferred is the sulfatide [ST-OH (18:1 _24:0)] having the structure:

According to the second aspect, the invention also relates to sulfatides for use in the diagnosis of a biliary tract disease, wherein the sulfatides are preferably selected from the group consisting of

[ST-OH (18:1 _ 24:0)], [ST-OH (18:1 1 6:0)],

[ST-OH (18:1 _ 24:1 )],

[ST-OH (18:1 _ 22:0)],

[ST(42:0)],

[ST(41 : 1 )],

[ST (42 : 1 ) ;

[ST (18:1 _ 16:0)] ,

[ST (18:1 _ 24 :0)] and

[ST (40:0)].

As described above, the method of the invention comprises determining the concentration of sulfatides in a biospecimen from the subject and comparing to a reference biospecimen. Different types of sulfatides can be measured, so the concentration of the individual sulfatide is determined and compared to the concentration of that particular sulfatide in the reference specimen. Measurement of concentrations is usually carried out using standards that can be added to the sample.

The difference in concentration of sulfatides between the subject biospecimen and the reference can be an increase in concentration or a decrease in concentration. Also, the types of sulfatides, i.e. the profile of the sulfatides can be different. Thus, in the biospecimen of the subject, one or more sulfatides can be abundant, wherease in the reference specimen other sulfatides can be abundant.

The most preferred sulfatide [ST-OH (1 8:1_24:0)] has been shown to be particularly indicative of biliary tract diseases.

Several methods are available to detect the sulfatides in the biospecimen. If the biospecimen is a tissue sample, in particular a tissue sample from liver, gall bladder or bile duct, the sulfatides are determined by a MALDI-MSI (Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging) method. MALDI- MSI allows to monitor the spatial distribution of bile salts and lipids in tissue.

The tissue section coated with matrix material can then be subjected to MALDI-MSI analysis. If the biospecimen is a biofluid, the sulfatide concentrations can be determined by known chromatography methods. Examples are U(H)PLC-MS (Ultra (High) Performance Liquid Chromatography) and U(H)PLC-MS/MS (Ultra (High) Performance Liquid Chromatography tandem Mass Spectometry). Antibody-based immunological assays are another method to quantify concentrations of total sulfatides.

A further method is to introduce a PET probe selective for the sulfatides.

Preferably the method of diagnosis of the invention is an in vitro method.

a) determining by means of a MALDI method the concentration of sulfatides in a biospecimen from the subject, wherein the biospecimen is a liver tissue;

b) comparing the concentration of sulfatides in the biospecimen from the subject with the concentration of sulfatides in a reference biospecimen;

wherein a difference of the concentration of the sulfatides in the biospecimen from the subject relative to the reference biospecimen indicates that the subject has an altered risk of having or developing PSC.

In another embodiment, the present invention provides for a method for diagnosing cholangiocarcinoma in a subject comprising the steps of :

wherein a difference of the concentration of the sulfatides in the biospecimen from the subject relative to the reference biospecimen indicates that the subject has an altered risk of having or developing cholangiocarcinoma.

Description of the drawings

Figures 1 A and 1 B show MALDI-IMS-MS of isomeric bile acids in negative ion mode. Mobilograms of (A) DCA/CDCA (m/z 391 .27) and (B) TDCA/TCDCA (m/z 498.28).

Figures 2A and 2B show multimodal imaging of healthy dog liver tissue at (A) 50 pm and (B) 15 pm spatial resolution. (I) Optical image of the tissue section prior to matrix application or staining. (I I) Tissue sections stained with hematoxylin and eosin stain and (I II) Masson’s trichrome. (A) Negative-ion MALDI-MS and (B) MALDI-FTICR-MS images showing the distribution of selected molecular species in the (IV) bile duct lumen ([TCA-H]- at m/z 514.2849); (V) connective tissue ([PA (18:0_1 8:1 )— H] at m/z 701 .5129); (VI) parenchyma ([PI (18:0_20:4)-H]^_ at m/z 885.5504); (VI I) bile duct ([ST-OH (18:1_24:0)-H] at m/z 906.6339) and (VI II) overlay of the selected species. Designated masses are from MALDI-FTICR-MS imaging measurements.

Figures 3A and 3B show multimodal imaging of healthy dog liver tissue at (A) 50 pm and (B) 15 pm spatial resolution. (I) Optical image of the tissue section prior to matrix application. (A) Positive ion mode MALDI-MS and (B) MALDI- FTICR-MS images showing the distribution of selected molecular species in the (II) bile duct lumen (unknown at m/z 576.2418), (III) blood vessels (heme [M]⁺ at m/z 616.1804), (IV) connective tissue ([PC (32:0)+K]⁺ at m/z 772.5254), (V) parenchyma ([PC (38:4)+K]⁺ at m/z 848.5564) and (VI) overlay of the selected species (masses observed in MALDI-FTICR-MS imaging measurements quoted).

Figures 4A and 4B show multimodal imaging of healthy human liver at (A) 50 pm and (B) 15 pm spatial resolution. (I) Optical image of tissue section prior to matrix application or staining. (II) Tissue sections stained with hematoxylin and eosin and (III) Masson’s trichrome stain. (A) Negative-ion MALDI-MS and (B) MALDI- FTICR-MS images showing the distribution of selected molecular species in the (IV) bile duct lumen ([TCA-H]^· at m/z 514.2849); (V) connective tissue ([PA (18:0_18:1 )-H] at m/z 701 .5128) ; (VI) parenchyma ([PI (18:0_20:4)-H]^_ at m/z 885.5504) and (VII) bile duct ([ST-OH (1 8:1_24:0)-H]- at m/z 906.6339). (VIII) Overlay of the selected species. Designated masses are from MALDI-FTICR-MS imaging measurements.

Figures 5A and 5B show multimodal imaging of healthy human liver tissue at (A) 50 pm and (B) 15 pm spatial resolution. (I) Optical image of tissue section prior to matrix application. (A) Positive ion mode MALDI-MS and (B) MALDI-FTICR- MS images showing the distribution of selected molecular species in the (II) bile duct lumen (unknown at m/z 560.2427), (III) blood vessels (heme [M]⁺ at m/z 616.1782), (IV) connective tissue ([PC (32:0)+K]⁺ at m/z 772.5254), (V) parenchyma ([PC (34:2)+K]⁺ at m/z 796.5252) and (VI) overlay of the selected species (masses observed in MALDI-FTICR-MS imaging measurements quoted).

Figure 6 shows a comparison of immunohistochemical staining of bile ducts, MALDI-MS imaging and TOF-SIMS imaging of (A) healthy human and (B) dog liver. (I) Hematoxylin and eosin stain of tissue section post-MALDI-MSI analysis. (II) Cytokeratin staining showing the bile duct epithelium. (Ill) MALDI-MS imaging and (IV) TOF-SIMS imaging of the bile ducts showing the distribution of [ST-OH (18:1_24:0)- H]- at m/z 906.634.

Figure 7 shows multimodal imaging of human liver from patients with

(A) mild and (B) advanced PSC. (I) Optical image of tissue section prior to matrix application or staining. Consecutive tissue sections stained with (II) hematoxylin and eosin (III) Masson’s trichrome. MALDI-FTICR-MS images showing the distribution of selected molecular species, namely (IV) [TCA-H]- at m/z 514.2855; (V) sulfated bile alcohol ([M-H]-) at m/z 531 .3004; (VI) bilirubin at m/z 583.2569; (VII) heme ([M-H]-) at m/z 615.1 704; (VI II) [PA (1 8:0_18:1 )-H] at m/z 701 .5128; (IX) [PI (18:0_20:4)-H]- at m/z 885.5504, and (X) [ST-OH (18:1_24:0)-H] at m/z 906.6353. (XI) Overlay of the selected species.

Figure 8 shows multimodal imaging of liver from a patient with PSC in positive ion mode at 15 pm spatial resolution. (A) Optical image of tissue section prior to matrix application. MS images showing the distribution of selected molecular species in the (B) bile duct lumen (unknown at m/z 560.2434), (C) heme ([M]⁺) at m/z 616.1804, (D) connective tissue ([PC (32:0)+K]- at m/z 772.5251 ), (E) parenchyma ([PC (34:2)+K]- at m/z 796.5254) and (F) overlay of the selected species (masses observed in MALDI-FTICR-MS imaging measurements quoted).

Figure 9 shows a comparison of immunohistochemical stain and MALDI-MS images from patient with advanced PSC. A) Optical image of tissue section prior to matrix application and B) cytokeratin staining of a consecutive tissue section. MALDI-MS images showing the distribution of C) [TCA-H]- at m/z 514.28, D) sulfated bile alcohol ([M-H]-) at m/z 531 .30, E) heme ([M-H]-) at m/z 615.17, F) [PA (18:0_1 8:1 )- H]- at m/z 701 .51 , G) [PI (18:0_20:4)-H]- at m/z 885.55, H) [ST-OH (18:1_24:0)-H]- at m/z 906.63 and I) bilirubin diglucuronide ([M-H]-) at m/z 935.32. J) Overlay of the selected species.

Figure 1 0 shows multimodal imaging of healthy rat liver in negative ion mode at 1 5 pm spatial resolution. (A) Optical image of tissue section prior to matrix application. MS images showing the distribution of selected molecular species in the

(B) bile duct lumen ([TCA-H]- at m/z 514.2845), (C) connective tissue ([PA (18:0_18:1 )- H]- at m/z 701 .5129), (D) parenchyma ([PI (18:0_20:4)-H]- at m/z 885.5499), (E) bile duct ([ST-OH (18:1_24:0)-H]- at m/z 906.6348) and (F) overlay of the selected species (masses observed in MALDI-FTICR-MS imaging measurements quoted). Figure 1 1 shows multimodal imaging of healthy rat liver in positive ion mode at 100 pm spatial resolution. (A) Optical image of tissue section prior to matrix application. MS images showing the distribution of selected molecular species in the (B) bile duct lumen (unknown at m/z 560.2414), (C) heme ([M]⁺) at m/z 61 6.1775, (D) connective tissue ([PC (32:0)+K]^_ at m/z 772.5254), (E) parenchyma ([PC (38:4)+K]⁺ at m/z 848.5566) and (F) overlay of the selected species (masses observed in MALDI- FTICR-MS imaging measurements quoted).

Figures 12A-12D show MALDI-MS/MS spectra obtained from the most abundant species. MALDI-MS/MS spectra obtained from 12A) bile duct lumen, 12B) connective tissue, 12C) parenchyma and 12D) bile duct.

Figure 13 shows MALDI-MS imaging of human cholangiocarcinoma liver tissue. (A) Optical image of tissue prior to matrix application. MALDI-MS images in negative ion mode showing the distribution of selected molecular species, viz. (B) the sulphated bile salt [GCDCA-S-H]- at m/z 528.26, (C) the connective tissue lipid marker [PS (18:0_1 8:1 )-H] at m/z 788.54, (D) the parenchymal lipid marker [PI (18:0_1 8:2)-H] at m/z 861 .55, (E) the bile duct lipid marker (hydroxylated sulfatide) [ST-OH (18:1 _24:0)-H] at m/z 906.64 and (F) overlay of the selected molecular species. Note that the tumors appear as dense clusters of sulfatide (e.g. in the middle part of the section), in panel E represented by the hydroxylated sulfatide C42:1 [ST- OH (18:1_24:0)-H] .

Figure 14 shows MALDI-MS imaging of liver tissue of another case of human cholangiocarcinoma, revealing co-localization of sulfatides with tumor regions. (I) General histology per staining with hematoxylin and eosin. MALDI-FT-ICR MS imaging in negative ion mode showing the distribution of selected molecular species in the (II) bile duct lumen (the bile salt [TCA-H]- at m/z 514.2849), (III) connective tissue (the lipid marker [PA (18:0_18:1 )-H]- at m/z 701 .5128), (IV) parenchyma (the lipid marker [PI (18:0_20:4)-H]- at m/z 885.5504), (V) and the bile duct (the lipid marker [ST-OH (18:1_24:0)-H]- at m/z 906.634) and (VI) overlay of the selected species.

Experimental

Introduction

During the past two decades, bile salts have been upgraded from molecules required for absorption of dietary lipids and fat-soluble vitamins, to signaling molecules regulating biological processes as diverse as nutrient metabolism, inflammation, and liver regeneration [1 -4] The discovery of bile salt receptors that mediate these signaling activities was key to the renewed interest in bile salts by academic and pharmaceutical communities. In particular, Farnesoid-X Receptor (FXR), a bile salt-activated transcription factor with an essential role in bile salt homeostasis, has been studied extensively. FXR agonism-based therapy has already been approved for treatment of patients with primary biliary cholangitis (PBC) and inadequate response to ursodeoxycholic acid [5]. Multiple clinical trials are ongoing to evaluate efficacy of FXR agonists in treatment of metabolic liver disorders like non alcoholic steatohepatitis (NASFI) [6-7]

The liver is the primary organ involved in the synthesis and handling of bile salts. Being amphipathic molecules, bile salts in the aqueous phase can interact with biological membranes. Consequently, the liver is the prime site where injury occurs when bile formation or flow of bile is impaired, and toxic levels of bile salts build up inside the parenchymal cells. At a certain threshold intracellular bile salts damage mitochondrial membranes and initiate a sterile inflammatory response that results in immune-mediated tissue injury [8-1 0]. In drug development, drug-induced cholestasis is a common reason for halting further development in pre-clinical phases of testing [1 1 ] ·

Distinct bile salt species arise from different synthetic routes in the host, utilization of either glycine or taurine for formation of A/-amidated conjugates, and biotransformation by the gut microbiota [1 ]. Individual bile salt species have distinct signaling properties and toxicity profiles. Mass spectrometry (MS)-based analytical tools have been of great value to determine bile salt composition of (homogenized) biospecimens (e.g. serum, bile, urine, feces, liver tissue) from healthy and diseased subjects.

Study of hepatic bile salt composition requires the preparation of a homogenate, inevitably resulting in loss of spatial information. As several aspects of hepatic bile salt handling are known to be zonated (e.g. extraction of bile salts from the portal supply in the periportal area, de novo synthesis in the pericentral area) [12], information on the local distribution of bile salts within the liver could further advance our understanding of bile salt (patho)physiology.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been extensively used to monitor the distribution of drugs, lipids, peptides and proteins in tissue sections, making this an ideal tool to determine the molecular profiles of the various compartments of healthy and diseased liver. Flere we used MALDI-MSI to study the spatial distribution of bile salts within the liver of animal species commonly used in pre-clinical drug testing, as well as in non- cholestatic and cholestatic human liver parenchyma. Specific aims were (i) to determine whether it is feasible to determine the spatial distribution of distinct bile salt species in the liver, including those with identical molecular mass, (ii) to identify lipid molecular markers that define the structural elements of the liver anatomy and (iii) determine if there are unique molecular profiles that can distinguish between animal species. This spatial molecular information can be employed to address roles of defined molecules in liver (dys)function, both in the context of human pathology and drug screening programs.

Materials

Cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), lithocholic acid (LCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), 2,5- dihydroxybenzoic acid (DHB) and 9-aminoacridine (9AA) were purchased from Sigma Aldrich (Zwijndrecht, The Netherlands). Chloroform (CHCI₃) and methanol (MeOH) was purchased from Biosolve (Valkenswaard, the Netherlands).

Liver specimens

A healthy canine liver specimen (Marshall Beagles, n=1 ) was obtained in the framework of an ongoing study at Janssen Pharmaceutica N. V. The study was approved by the local animal ethical committee and conducted in facilities accredited by national institutions adhering to AAALAC guidelines. Human liver specimens were obtained from patients undergoing either resection for hepatobiliary malignancies (non-affected liver tissue sample distant from the tumor, n=1 ) or undergoing liver transplantation for end-stage liver disease (PSC, n=2). Specimens were procured as part of biobank initiatives at the Aachen (Medical Ethical approval #EK 20609) and Maastricht (Medical Ethical approval #14-4-153) locations of the Euregional Surgical Center Aachen Maastricht (ESCAM). Patients gave written informed consent for biobanking and research use of surgical specimens. A rat liver specimen (Wistar Han, n=1 ) was obtained from a control group of rats sacrificed for a study on colonic anastomotic healing (approved by the local Animal Ethics Committee of the Radboud University, Nijmegen). Per liver specimen, multiple sections throughout the tissue were analyzed, i.e. dog (n=7), non-cholestatic patient (n=7), PSC patients (n=4) and rat (n=9).

Tissue preparation

Liver tissue was sectioned using a Microm HM535 cryo-microtome (Microm International, Walldorf, Germany) at a temperature of -20 °C to produce 10 pm-thick sections, which were thaw-mounted onto clean indium tin oxide (ITO)-coated glass slides (4-8 W resistance, Delta Technologies, Stillwater, MN, USA). Tissue sections were scanned prior to matrix application using a Nikon Super CoolScan 5000 ED (Nikon Corporation, Tokyo, Japan) to produce high-quality optical images. Consecutive tissue sections were thaw-mounted on regular glass slides (Thermo Scientific) for histological and immunohistochemical staining.

Matrix application

Tissue sections were coated with 15 mg/mL DHB in 2:1 CHCLiMeOH with 0.2% TFA using the SunCollect automated pneumatic sprayer equipped with dispenser system (Sunchrom GmbH, Friedrichsdorf, Germany) in a series of 15 layers. The initial seeding layer was performed at 1 0 pL/min; layers were performed at 20, 30, and 40 pL/min, respectively (all at speed 450 mm/min, track spacing: 2 mm, height: 23 mm). For negative-ion mode some tissue sections were coated with 10 mg/mL 9AA in 70:30 MeOH:H₂0 in a series of 10 layers. The initial seeding layer was performed at 10 pL/min; layers were performed at 1 5, 20, and 25 pL/min, respectively (all at speed: 450 mm/min, track spacing: 2 mm, height: 25 mm).

Instrumentation

MALDI-MSI. High-speed imaging was performed on a Bruker RapifleX MALDI Tissuetyper™ system (Bruker Daltonik GmbH, Bremen, Germany), a more detailed description of this instrument is provided elsewhere [1 5]. The instrument was operated in reflectron mode in both positive- and negative-ion modes in the mass range m/z 400-1 000. The instrument was calibrated prior to analysis using red phosphorus clusters in both polarities. Images of the whole tissue sections were acquired using a 50 50 pm raster (25 25 pm beam scan area). The images were generated using the Fleximaging version 5.0 software (Bruker Daltonik GmbH) and were normalized to the total ion current (TIC). MALDI-FTICR-MSI. For confirmation of molecular identity, high-mass resolution (100,000 at m/z 500) measurements were performed on a Bruker SolariX FTICR mass spectrometer (Bruker Daltonik GmbFI, Bremen, Germany) equipped with a 9.4 T superconducting magnet. The instrument was operated in both positive- and negative-ion modes in the mass range m/z 100-2000. The instrument was calibrated prior to analysis using red phosphorus clusters in both polarities. Images were acquired with a pixel size of 15 pm. A 95% data reduced profile spectrum and spectrum peak list were saved for further analysis.

MALDI-MS/MS. For further confirmation of molecular identity, a Waters MALDI FIDMS Synapt G2-Si mass spectrometer (Waters Corporation, Manchester, UK) was used to acquire IMS-MS and tandem mass spectrometry (MS/MS) spectra. The instrument was calibrated using red phosphorus prior to analysis. The ion of interest was fragmented by collision induced dissociation (CID) in the trap cell with an isolation window of 1 Da. For presentation purposes, mass spectra from the MassLynx version 4.1 software (Waters Corporation) were exported in the form of text files and imported into mMass, open-source software used for mass spectral processing [16]. The MS/MS spectra from the commercial bile acid standards and tissue sections were compared to those in the literature or databases such as lipid maps (www.liDidmaps.org) and the ALEX123 lipid calculator (www.alex123.info). The fragment ions observed in each MS/MS spectrum were assigned based on the recently proposed nomenclature for lipids [17]

TOF-SIMS. High spatial resolution imaging was performed using a PHI nanoTOF II TOF-SIMS instrument (Physical Electronics, Minnesota, MN, USA) equipped with a 30 kV Bi_n ^q+ cluster liquid metal ion gun (LMIG), which was operated with a Bi³⁺ cluster ion beam. Images were acquired using a tile size of 500 pm at 30 frames with an ion dose of 2.59 c 10¹² ions/cm² in negative ion mode, the data was acquired in the mass range m/z 0-1850. The images were generated using software PHI TOF-DR software (Physical Electronics) [18].

Tissue staining

Cryosections (5-pm thick) of liver tissue were used for immunohistochemistry. Flematoxylin- and eosin (H&E) and Masson’s trichrome (MTC) staining was performed on consecutive tissue sections followed by aqueous mounting. Optical images of all stained tissue sections were acquired using a Mirax desk scanner (Zeiss, Gottingen, Germany). The bile duct epithelium was stained using a wide spectrum anti-cytokeratin antibody (Dako/Agilent Pathology Solutions) and secondary detection reagents.

Statistical analysis

Statistical analyses were performed on the MALDI-FTICR-MSI data using the in-house built ChemomeTricks toolbox for Matlab version 7.0 software (The MathWorks, Natick, MA, USA) [19]. Briefly, peak picking was performed on an average spectrum (created using the peaks sqlite file) at a 0.001 Da bin size. The center of the peaks and integration ranges were defined, to account for peak shifting from pixel- to-pixel. Principal component analysis (PCA) was then performed on the converted datasets, which were normalized with the total ion current. The peak list from each principal component that highlighted different areas of the liver were exported and the top ten peaks (based on intensity) were then identified. Results and Discussion

Identification of bile salts

In order to have optimal sensitivity and selectivity of the method applied, the physiologically most relevant (non)conjugated primary and secondary bile salts (see Table 1 below)., were spotted individually on a target plate, coated with 9AA and analyzed with MALDI-MS, according to previous work [20, 21 ] All bile salts were observed in their deprotonated form ([M-H] ). MS/MS experiments were performed on the standards (see Table 1 below).

A

B

Name R1 R2 R3 R4

CA H a-OH OH OH

CDCA H a-OH H OH

LCA H H H OH

DCA H H OH OH

C

Name Type Formula Mass ((M-H]^~)

CA Primary C24H35O5 40 .26

CDCA Primary C₂₄H₃,0₄ 391.26

LCA Secondary C₂₄H₃₉OJ 375.27

DCA Secondary CyHssAi 391.26

TCA Primary C₂₆H₄₄N0₇S 514.28

TCDCA Primary C^NOeS 498.28

TDCA Secondary C₂₆H₄₄N0₆S 498.28

GCA Primary C₂₄H₄₂N0₆ 464 30

GCDCA Primary C_2{H₄₂NO_S 448.30

GDCA Secondary C₂₆H₄₂NOJ 448.30

In Table 1 , A) represents the base structure of bile acids and B) the positions of the hydroxyl groups and site of conjugation. C) represents the observed masses of bile acid standards in negative ion mode.

The taurine-conjugated bile salts showed characteristic product ions, indicating the fragmentation or loss of taurine such as m/z 80 (S0₃ ), 107 (C₂H₃O₃S ) and 124 (C H NO S ), whereas the glycine-conjugated bile salts showed the loss of glycine m/z 74 (C₂H₅NO₂ ) [22, 23]. The unconjugated bile salts showed fragments resulting from the loss of H 0, C0 and CH 0 [23]. Isomeric species, such as chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) and their conjugated forms, can be separated using chromatographic techniques [23]. Here, ion mobility separation (IMS) was evaluated for separation of isomeric bile salts. Standards of the isomeric bile salts were applied as a mixture on a target plate and analyzed. Using this technique it was possible to differentiate between DCA and CDCA (Figure 1 A), however it was not possible to separate the taurine-conjugated variants thereof (Figure 1 B). This is likely due to the limited resolution of current commercial ion mobility instrumentation. Since the bulk of hepatic bile salts are conjugated, IMS was not suitable to analyze isomeric bile salt species in intact tissues.

Spatial distribution of bile salts and lipids in healthy dog liver.

High-speed imaging experiments on whole liver tissue sections (at a spatial resolution of 50 pm) showed the distinct distribution of various molecular markers linked to the different tissue types found in the liver. Regions such as the parenchyma, the bile ducts, the connective tissue and the bile duct lumen could be clearly distinguished in these MALDI-MS images (Figure 2A and 3A). Following analysis of the whole tissue section, high mass and high spatial resolution MALDI- FTICR-MSI was performed on specific areas, selected based on histological and molecular features (Figure 2B and 3B). These images provided greater spatial detail of the selected molecular species and improved mass accuracy, improving molecular identification.

MALDI-MSI in negative-ion mode showed the presence of predominantly taurine-conjugated bile salts, the most abundant of was taurocholic acid (TCA) and taurochenodeoxycholic/taurodeoxycholic acid (TCDCA/TDCA). This is to be expected as dogs use primarily taurine for bile acid conjugation [25, 26]. The distribution of TCA at m/z 514.28 (Figure 2A, IV) showed localized patches of varying sizes throughout the tissue, most likely representing the bile ducts. The larger ones (average diameter 1 75-250 pm), which are in close proximity to the large blood vessels, are septal bile ducts. Smaller bile ducts (average diameter 20-70 pm) distribted throughout the tissue, are the interlobular bile ducts [27] Interestingly, 3- keto-TCA and 3-keto-TDCA/TCDCA were also present in the bile duct lumen. Keto bile salts were previously detected in bear bile [28]. Next to this, taurolithocholic acid was also present. The connective tissue showed a strong presence of a lipid at m/z 701 .51 (Figure 2A, V), which was identified as a phosphatidic acid [PA (18:0_18:1 )— H] . This species was distributed throughout the tissue but appeared to be more abundant in the connective tissue, which correlates well with Masson’s trichrome staining. The connective tissue was also characterized by other phosphatidic acids, phosphatidylserines (PS) and sphingomyelin (SM) fragments ([M-CFE] ) [29]. It should be noted that the positive ion counterparts of the SM fragments co-localized with observed species, hence confirming their identification (See Figure 3A/3B). In the surrounding liver parenchyma, the most abundant species, which was homogenously distributed throughout the parenchyma but absent from the other areas of the tissue, was identified as a phosphatidylinositol (PI), specifically [PI (1 8:0_20:4)-H] at m/z 885.55 (Figure 2A, VI). Other Pis, PAs and phosphatidylethanolamines (PE) were predominantly present in this area.

Another lipid species at m/z 906.64 (Figure 2A, VI I) was distributed in small localized patches throughout the tissue, and co-localized with the distribution of bile acids. MALDI-FTICR-MS analysis, with higher mass and higher spatial resolution capabilities, revealed that the species at m/z 906.6339 was localized in a thin lining on the inside of the bile duct (Figure 2B, VI I). This compound was identified, by MS/MS measurements, as a hydroxyl-sulfatide [ST-OFI (1 8:1_24:0)-H] , a ceramide derivative previously identified by MALDI-MS/MS imaging of brain tissue [29]. Non-hydroxylated sulfatides (ST) were also present in the bile duct. The overlay of the selected molecular species (Figure 2B, VII I) recapitulated the general histological features of the liver. All peaks identified in the different regions of the healthy dog liver are shown in Tables 2 and 3 below .

Spatial distribution of bile salts and lipids in healthy human liver.

Healthy human liver tissue was analyzed next to study (dis)similarities in spatial distribution of bile salt species, and molecular lipid markers of structural elements in the human liver (Figure 4A and 4B and Figures 5A and 5B). MALDI-MSI showed high signal intensities of TCA at m/z 514.28 and TCDCA at m/z 498.28, whereas lower signal intensities of glycochenodeoxycholic acid (GCDCA) at m/z 448.30 and glycocholic acid (GCA) at m/z 464.30 were present (Table 4). Table 4 Serum biochemistry form the two PSC patients

Diagnosis ALT AST Bilirubin ALP GGT INR

[U/L] [U/L] [mg/dL] [U/L] [U/L]

PSC 1 15 19 0.90 N/A 21 1 .05

PSC 2 76 79 2.81 365 299 1 .1 1

Key: Alanine aminotransferase (ALT) , aspartate aminotransferase (AST), alkaline phosphatase (ALP) , gamma glutamyltransferase (GGT), interantional normalized ratio (I N R). These findings were somewhat unexpected since the human bile salt pool consists largely of glycine-conjugated species, with taurine-conjugated bile salts comprising approximately 20% of the biliary bile salt pool [26]. This discrepancy is most likely attributed to the fact that the sulfonate group of taurine conjugates is more easily ionized in negative-ion mode than the carboxylic acid group of glycine conjugates [20, 21 ] The distribution of TCA at m/z 514.28 (Figure 4A, IV) showed a few small localized patches throughout the tissue, indicating the presence of bile ducts. Both septal and interlobular bile ducts were apparent in the studied specimen. Taurine-conjugated keto bile salts and lithocholate were also detected in the bile ducts Also in human liver tissue, the connective tissue was defined by [PA (18:0_18:1 )— Hj at m/z 701 .51 (Figure 4A, V), and distribution of this lipid species matched the Masson’s trichrome staining. Connective tissue was also characterized by SM fragments and PS. Fluman liver parenchyma (Figure 4A, VI) was characterized by a PI [PI (18:0_20:4)— Hj at m/z 885.55, as well as other Pis, PAs and PEs. The hydroxyl- sulfatide [ST-OFI (18:1_24:0)— H] at m/z 906.63 (Figure 4A, VII) appeared as small localized patches throughout the tissue. MALDI-FTICR-MSI analysis showed the hydroxyl-sulfatide [ST-OFI (18:1_24:0)— H] at m/z 906.6340 as a thin band located in the bile duct (Figure 4B, VII). The overlay of the selected species (Figure 4B, VIII) demonstrated the localization of the bile salts within the bile duct lumen, which was bordered by a lining of the aforementioned hydroxyl-sulfatide. Additional hydroxylated and non-hydroxylated sulfatide species were observed in the bile duct wall. The peaks identified in the different regions of the healthy human liver are shown in Tables 2 and 3 below.

Sulfatides are novel molecular markers for the bile duct epithelium.

Hydroxyl-sulfatide [ST-OFI (18:1_24:0)— H]- was identified in both healthy dog and human liver tissue, and specifically localized as a thin band lining the bile duct. This compound was the most prominent of several hydroxylated and non- hydroxylated sulfatides that colocalized in the bile duct (Table 3). In order to determine whether sulfatides are specific markers for the bile duct, immunohistochemical staining for cytokeratins was performed on sections of dog and human liver (Figure 6). This revealed the presence of cytokeratins in the cholangiocytes/bile duct epithelium (Figure 6, II). MALDI-MSI of a consecutive section revealed [ST-OFI (18:1_24:0)-H] at m/z 906.634 distributed as a ring-like structure (Figure 6, III) that enclosed luminal bile salts. High spatial resolution TOF-SIMS imaging of consecutive sections of dog and human liver confirmed the confinement of sulfatides to the bile duct epithelium (Figure 6, IV). The molecular images correlate well with the immunohistochemical staining of the bile ducts indicating that this species, as well as the other sulfatide species (Table 3), are molecular markers for the bile duct epithelium.

The unique localization of sulfatides to bile duct epithelium suggests a biologically relevant role. Sulfatides are glycosphingolipids that contain an extra sulfate group and hence are commonly observed in negative-ion mode. Sulfatides are predominantly found in the exoleaflet of the membrane bilayer, and have been detected in different tissues, including the brain where they are an abundant component of the myelin sheath, the pancreas in the islets of Langerhans, and the kidney [30]. Although sulfatides were previously detected in liver, their presence could not be attributed to a particular region because those studies were performed on tissue extracts [31 ]. Sulfatides are negatively charged and will likely repel negatively charged compounds, such as bile salts, and may accordingly play a role in protecting the bile duct epithelium from detergent action of bile salt anions. Information on specific roles of sulfatides in liver biology is sparse. Sulfatides are self-lipid antigens, and presence of autoantibodies appears to be common in chronic autoimmune liver diseases like autoimmune hepatitis and primary biliary cholangitis (PBC) [32] Sulfatides presented by CD1 b protein are recognized by type 2 natural killer T (NKT) cells. In the liver, NKT cells are abundant in the sinusoids, with type I and type II NKT cells having a pro- and anti-inflammatory action, respectively [33]. Immune-mediated liver injury in an animal model of alcoholic liver disease is dependent on type I NKT cells [34] Interestingly, activation of type II NKT cells by sulfatides prevented inflammation and liver disease in this model, and may have a broader role in a tolerogenic response in inflammatory liver disease [35]. Given the abundance of this lipid in the bile ducts, a link with inflammatory cholangiopathies like PBC and primary sclerosing cholangitis (PSC) is plausible. Spatial distribution of bile salts and sulfatides in liver of PSC patients

Liver tissue from initially two patients undergoing liver transplantation for treatment of PSC was analyzed by MALDI-FTICR-MSI (Figures 7A, 7B and 8) to explore whether molecular species are globally similar in diseased liver tissue. The first PSC patient (mild PSC) had normal liver tests, with no indications for cholestatic liver injury (Table 4). Histological evaluation of a liver biopsy taken prior to transplantation revealed mild inflammation but no fibrosis. The second PSC (advanced PSC) had liver test abnormalities indicating cholestasis and liver injury (Table 4).

Mild PSC. Hematoxylin and eosin (Figure 7A, II) and Masson’s trichrome staining of liver sections of the patient with mild PSC (Figure 7A, III) revealed concentric periductal fibrosis with little periductal inflammatory infiltrate. In addition, mixed inflammatory cells and bile ductular proliferation were found at the periphery of the portal area extending into the interlobular septa. For both patients the histological features observed in the hepatic parenchyma, bile ducts and portal areas are associated with PSC [36]. Despite these morphological differences, liver tissue of the patient with mild PSC showed the same distribution of selected molecular species as healthy human liver tissue. Taurine-conjugated CDCA/DCA species were the most abundant bile salt species, and localized to the bile duct lumen (Figure 7A, IV). Molecular markers identified in the healthy human liver also defined the connective tissue, parenchyma and the bile ducts in the patient with mild PSC (Figure 7A). The distribution of [ST-OH (1 8:1_24:0)-H] at m/z 906.6353 (Figure 7A, X) indicated that the bile ducts were narrowed, a hallmark histological feature observed in PSC [37] Blood vessels in the portal area were marked by the presence of heme (Figure 7A, VII).

Advanced PSC. Histological examination of the liver from the patient with advanced PSC (Figure 7B, II and III) showed a diminished number/absence of interlobular bile ducts, portal-to-portal bridging fibrosis, bile ductular proliferation in portal areas, and fibrous septa with minimal mononuclear inflammatory infiltrate. In addition nodular hepatocellular regeneration, ballooning degeneration of hepatocytes, and yellow to brown pigmentation were noted. Taurine-conjugated CDCA/DCA species were also present in the liver of the patient with advanced PSC (Figure 7B, IV), however they appear to have diffused out of the bile ducts into the surrounding parenchyma. This is reminiscent of regurgitation of bile salts from leaky bile ducts into the portal tracts in an animal model of PSC [38]. Moreover, a unique bile salt species at m/z 531 .2999 was observed (Figure 7B, V). MS/MS measurements indicated loss of m/z 97 (HS0₄ ) and m/z 80 (S0₃ ), leading to its tentative identification as the sulfated bile alcohol, 5-cyprinol sulfate. To our knowledge, the presence of 5-cyprinol sulfate (a sulfated bile alcohol abundant in cypriniform fishes) [39], has not been previously reported in humans, and likely relates to cholestasis where elevation of urinary bile alcohols is observed [40].

In order to confirm molecular identity, a whole-body tissue section of a zebra fish, an authentic source of 5-cyprinol sulfate, was analysed. The MALDI- MS/MS spectrum of 5-cyprinol sulfate obtained from the initial patient with severe PSC was comparable to that obtained from the zebra fish.

Glycochenodeoxycholic acid-sulfate (G(C)DCA-S) at m/z 528.2639 was also identified in the advanced PSC liver. In general, sulfation of bile salts is an adaptive mechanism to promote elimination of bile salts that have accumulated in the liver [41 ]

Furthermore, both unconjugated and conjugated bilirubin were observed. The distribution of bilirubin at m/z 583.2569, bilirubin monoglucuronide at m/z 759.2899 and bilirubin diglucuronide at m/z 935.3218 (Figure 7B, VI) coincided with that of bile salts. Of note, bilirubin (conjugated and unconjugated) was not detected in the liver from the patient with mild PSC and serum bilirubin levels within the normal range. Elevation of bilirubin can be seen following intrahepatic obstruction of bile flow resulting in the release of conjugated bilirubin into the bloodstream. The lipid at m/z 701 .5128, identified as [PA (18:0_18:1 )-H] was abundant in the connective tissue surrounding the blood vessels and bile ducts, with a notably thicker layer when compared to liver of the patient with mild PSC (Figure 7A, VII I). This is in line with extensive fibrosis throughout the tissue section in advanced PSC (Figure 7B, VIII). Despite the occurrence of discrete patches of biliary constituents (i.e. bile salts and bilirubin glucuronides) that indicates that bile ducts are present, hydroxylated and non- hydroxylated sulfatides were virtually absent in the liver of the patient with advanced PSC (Figure 7A, X and Figure 9). This could be due to the immune-mediated destruction of larger bile ducts at later stages of the disease. It is unclear if this is a general feature in advanced PSC and whether its absence/disappearance has pathological relevance.

To determine the generality of the above findings, liver samples from four additional patients with PSC were analysed and confirmed the presence and localization of 5-cyprinol sulfate and glycochenodeoxycholic acid-sulfate (G(C)DCA-S in each sample, albeit in varying abundance. !ntersoecies comparison of molecular profiles of healthy livers

Healthy rat liver was analyzed in the same manner, the resulting MALDI-FTICR-MS images are shown in Figures 10 and 1 1 . A distinct feature of the rodent liver is the synthesis of muricholic acids, that along with TCA are major bile salts in rodent bile. These tri-hydroxy bile salt species (and taurine-conjugated versions thereof) have an identical molecular mass as cholic acid (and taurine- conjugated versions thereof), and these isomeric species cannot be discriminated using current IMS equipment. Hence, it is most likely that the signal observed at m/z 514.28 corresponds to multiple taurine-conjugated tri-hydroxy bile salts. The most abundant species observed in the connective tissue, parenchyma and bile duct of healthy dog and human liver, were also observed in healthy rat liver (Table 3).

PCA was performed on the MALDI-FTICR-MSI datasets of the healthy liver specimens in order to determine the molecular profiles of each liver region in the animal species studied [42] The top 10 of identified lipid classes were determined for the positive and negative ion mode datasets (Tables 2 and 3, respectively) and classified by the following liver tissue structures: bile duct lumen, bile duct, connective tissue, and parenchyma (Table 5).

Table 5: Summary of lipid species detected in healthy and cholestatic liver tissue

Positive ion mode

Negative ion mode

Note: Species in positive ion mode were observed as protonated ([M+H]+), sodium ([M+Na]+) and potassium ([M+K]+) adducts. Negative ion species were observed exclusively as deprotonated ( [M- H]-) species.

† No species related to the bile duct were detected in positive ion mode. f U nknown species present in the bile duct lumen (identification currently in progress).

Key: Bile acids (BA), Phosphatidic acid (PA), Phosphatidylethanolamine ( PE), Phosphatidylserine ( PS), Phosphatidylinositol (P I), Phosphatidylcholine ( PC), Sphingomyelin (SM), Sulfatide (ST) and Hydroxyl-Sulfatide (ST-OH) .

Localization of the most abundant lipid species for each of these regions is also depicted in Figures 2, 4 and 7. The MALDI-MS/MS spectra obtained directly from liver tissue sections of the most abundant molecular markers in each region of the liver are presented in Figures 12A, 12B, 12C and 12D. The same molecular classes were observed in the examined structures across all species, the only difference was the pattern of bile salt conjugation. In human and mild PSC liver tissue, both glycine- and taurine-conjugated bile salt species were detected, whereas only taurine-conjugated bile salts were apparent in dog and rat liver. Interestingly, in the advanced PSC liver taurine conjugated bile salts, bilirubin (conjugated/un conjugated) and sulfated bile salts/alcohols were present. Negative ion mode was far more informative than positive ion mode, as the bile salts and a wide range of lipids are more easily detected in negative ion mode. In addition, the mass spectrum is less complicated as only deprotonated ([M-H] ) species are present, whereas in positive ion mode the spectrum is compounded by protonated species ([M+H]⁺) and adducts of sodium ([M+Na]⁺) and potassium ([M+K]⁺). The positive ion MALDI-FTICR-MS images from healthy dog and human liver, as well as diseased human liver are shown in Figures 3, 5 and 8 respectively. In positive ion mode, a number of yet unknown molecular species (ranging from m/z 544.24 - 592.24) separated by 16 Da, were detected in the bile duct lumen (data not shown). No specific molecular species were observed in the bile duct epithelium in positive ion mode.

Using MALDI-MSI, we identified lipid-specific distributions in different compartments of the liver in all three species (rat, dog, human), in both positive and negative ion mode. In the studied liver specimens, bile salts were largely confined within the biliary lumen. It will be interesting to examine the spatial distribution of the distinct bile salt species in the setting of bile salt retention as consequence of intrahepatic cholestasis. (Hydroxyl)-sulfatides were identified as specific molecular marker for the bile ducts and localized uniquely to bile duct epithelium. Experimental links with inflammatory events, warrants exploration of a possible involvement of sulfatides in inflammatory cholangiopathies like PBC and PSC. Spatial distribution of bile salts and sulfatides in cholangiocarcinoma liver tissue MALDI-MS imaging of human cholangiocarcinoma liver tissue is shown in Figure 13. The tumors appear as dense clusters of sulfatide (e.g. in the middle part of the section), in panel E in Figure 13 represented by the hydroxylated sulfatide C42:1 [ST- OH (18:1_24:0)-H] .

o o

Table 3 (part I)

oc e

O

H

W

o o

O b4

O

Table 3 (part II)

-4

n H bo o o

-

-4

Table 3 (part III)

O b4

O

Table 3 (part IV)

-4 *

n H bo o o

Table 3 (Part V)

Notes to Table 3: Species in positive ion mode were observed at protonated ([M+H]+_, sodium ([M+Na]+) and potassium ([M+K]+) adducts. Negative ion species were observed exclusively as deprotonated ([M- H]-) species.

Fatty acid composition of positive ions could only be obtained from MS/MS of protonated species. Key: Bile acids (BA), Phosphatidic acid (PA) , Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phospatidylinositol (PI), Phosphatidylcholine (PC), Sphingomyelin (SM) and Hydroxyl-sulfatide (ST-OH).

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Claims

1 . An in vitro method for diagnosing a biliary tract disease in a subject comprising the steps of :

b) comparing the concentration of the one or more biomarkers in the

biospecimen from the subject with the concentration of the one or more biomarkers in a reference biospecimen;

c) diagnosing the subject with the biliary tract disease if the concentration of the one or more biomarkers in the biospecimen from the subject is different from the concentration of the one or more biomarkers in the reference biospecimen wherein the one or more biomarkers are selected from the groups consisting of sulfatides and 5-cyprinol sulfate.

2. The method according to claim 1 , wherein the biliary tract disease is selected from the group consisting of primary biliary cholangitis, primary sclerosing cholangitis (PSC), intrahepatic, perihilar and distal cholangiocarcinoma, cholecystitis, choledocholithiasis and gallbladder cancer.

3. The method according to claim 1 or 2, wherein the biospecimen is a tissue

sample, preferably a tissue sample from liver, gall bladder or bile duct.

4. The method according to claim 3, wherein the biospecimen is a tissue sample from liver, gall bladder or bile duct and wherein the one or more biomarkers are determined by a MALDI method.

5. The method according to claim 4 for diagnosing primary sclerosing cholangitis (PSC) in a subject comprising the steps of:

a) determining by means of a MALDI method the concentration of the one or more biomarkers in a biospecimen from the subject, wherein the biospecimen is a liver tissue;

b) comparing the concentration of the one or more biomarkers in the

biospecimen from the subject with the concentration of the one or more biomarkers in a reference biospecimen; wherein a difference of the concentration of the one or more biomarkers in the biospecimen from the subject relative to the reference biospecimen indicates that the subject has an altered risk of having or developing PSC.

6. The method according to claim 4 for diagnosing cholangiocarcinoma in a subject comprising the steps of :

7. The method according to claim 1 or 2, wherein the biospecimen is a biofluid, preferably blood plasma, blood serum, bile, urine, luminal contents of the intestines or faeces.

8. The method according to claim 7, wherein the biospecimen is a biofluid and wherein the one or more biomarkers are determined by U(H)PLC-MS and U(H)PLC-MS/MS.

9. The method according to any one of claims 1 to 8, wherein the biomarker is 5- cyprinol sulfate.

10. The method according to claim 9, wherein the biliary tract disease is primary sclerosing cholangitis (PSC).

1 1 . The method according to any one of claims 1 to 8, wherein the one or more biomarkers are sulfatides, preferably selected from the group consisting of [ST-OH (18:1 _ 24:0)],

[ST-OH (18:1 _ 16:0)] ,

[ST-OH (18:1 _ 24:1 )],

[ST-OH (18:1 _ 22:0)],

[ST(42:0)j,

[ST(41 :1 )], [ST(42:1 ),

[ST (18:1 _ 16:0)],

[ST (18:1 _ 24 :0)] and

[ST (40:0)].

12. The method according to claim 1 1 , wherein the sulfatides are selected from [ST-OH (18:1 24:0)],

[ST-OH (18:1 _ 16:0)] ,

[ST-OH (18:1 _ 24:1 )] ; and

[ST-OH (18:1 _22:0)].

13. The method according to claim 12, wherein the sulfatide is [ST-OH

(18:1 24:0)].

14. The method according to any one of claims 1 1 to 13, wherein the sulfatides are detected in the bile duct.

15. The method according to any one of claims 1 1 to 14, wherein the biliary tract disease is primary sclerosing cholangitis (PSC), perihilar or distal

cholangiocarcinoma.

16. 5-Cyprinol sulfate for use in the diagnosis of a biliary tract disease.

17. Sulfatides for use in the diagnosis of a biliary tract disease, wherein the

sulfatides are preferably selected from the group consisting of

[ST-OH (18:1 24:0)],

[ST-OH (18:1 _ 16:0)] ,

[ST-OH (18:1 _ 24:1 )],

[ST-OH (18:1 22:0)],

[ST(42:0)],

[ST(41 :1 )],

[ST(42:1 ) ;

[ST (18:1 _ 16:0)],

[ST (18:1 24:0)] and

[ST (40:0)].

1 8. A kit for the diagnosis of a biliary tract disease, wherein the kit contains

• means for collecting a biospecimen ;

• means for measuring 5-cyprinol sulfate or sulfatides in the biospecimen ;

• optionally, instructions for using the kit;

• optionally, a reference sample.