CN113588802B

CN113588802B - Biomarkers and methods for non-invasive detection of liver toxicity pyrrolizidine alkaloid exposure

Info

Publication number: CN113588802B
Application number: CN202110254982.7A
Authority: CN
Inventors: 林革; 祝琳; 马江; 张淳瑗; 贺益盛
Original assignee: Chinese University of Hong Kong CUHK
Current assignee: Chinese University of Hong Kong CUHK
Priority date: 2020-03-10
Filing date: 2021-03-09
Publication date: 2024-03-19
Anticipated expiration: 2041-03-09
Also published as: WO2021180086A1; CN113588802A

Abstract

Described herein are non-invasive methods and kits for detecting, diagnosing, or monitoring pyrrolizidine alkaloid exposure in a subject by measuring the amount or concentration of a pyrrole-amino acid adduct in a biological sample (e.g., urine) from the subject, and by comparing the amount or concentration of the pyrrole-amino acid adduct to a reference control subject.

Description

Biomarkers and methods for non-invasive detection of liver toxicity pyrrolizidine alkaloid exposure

Cross Reference to Related Applications

The present application claims the benefit and priority of U.S. provisional application No. 62/987,769, filed on even 10/03/2020, the entire contents of which are hereby incorporated by reference herein.

Technical Field

The present application relates to biomarkers of pyrrolizidine alkaloid exposure to hepatotoxicity and methods for non-invasively detecting, diagnosing, monitoring and treating pyrrolizidine alkaloid exposure and diseases and conditions associated therewith.

Background

Pyrrolizidine alkaloids (pyrrolizidine alkaloid, PA) are phytotoxins produced by over 6,000 plant species of a variety of different families such as lithospermaceae, asteraceae, orchidaceae, leguminosae, convolvulaceae and Gramineae. About 500-600 potentially toxic PA and PA N-oxide have been identified in about 3-5% of the flowering plants in the world. Humans are easily exposed to toxic PA by eating herbal, herbal tea and dietary supplements made from PA-containing plants and/or PA-contaminated staple foods such as wheat and millet. In addition, the transfer of PA from livestock to dietary foods such as milk, eggs, meat, and honey through the food chain significantly increases the chance of PA exposure to the population. PA is therefore one of the most common types of natural toxins affecting humans, domestic animals and wild animals.

Intake of high doses of PA causes acute hepatotoxicity, in particular accompanied by severe liver injury known as liver sinus occlusion syndrome (HSOS), characterized by hepatomegaly, jaundice, ascites, hemorrhagic necrosis and hepatic vein occlusion. Acute hepatotoxicity is generally not apparent with prolonged exposure to relatively low levels of PA, but latent chronic diseases such as liver fibrosis, cirrhosis and cancer may develop progressively. The first PA poisoning of humans was reported in 1920.

Most reported PA poisoning cases are diagnosed based on obvious clinical symptoms and a confirmed history of taking PA-containing herbs or PA-contaminated foods. However, without a specific test for PA exposure, the number of cases of actual acute PA poisoning may be much higher than the number of cases currently recorded. Furthermore, due to the lack of specific and definitive diagnostic methods, determining whether PA exposure directly causes, indirectly causes or exacerbates liver injury and chronic liver disease is extremely challenging and results unsatisfactory. At present, it is practically impossible to rapidly confirm the etiological and confirmatory diagnosis of PA exposure in patients suspected of having hepatotoxicity, and to provide appropriate therapeutic and prophylactic measures for those patients. Thus, there is a need to develop specific biomarkers of PA exposure and non-invasive assay methods for qualitative and quantitative measurement of these specific biomarkers in mammals, particularly humans. The present invention meets these and other related needs.

Disclosure of Invention

In one aspect, the invention provides a method for detecting pyrrolizidine alkaloid exposure in a subject of interest. The method comprises the following steps: (i) Measuring the concentration or amount of pyrrole-amino acid adducts in a biological sample obtained from the target subject; (ii) Comparing the measured concentration or amount of the pyrrole-amino acid adduct to the concentration or amount of a reference control; and (iii) deeming detection of pyrrolizidine alkaloid exposure in the target subject when the measured concentration or amount of pyrrole-amino acid adduct in the sample is higher than the concentration or amount of the reference control. In some embodiments, the biological sample obtained from the target subject and used in any of the methods described herein is a urine sample.

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound according to formula I:

or an isomer thereof, wherein:

x and Y are independently selected from-OH and an amino acid, provided that at least one of X and Y is an amino acid; and

each amino acid is independently selected from the group consisting of histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan, and tyrosine.

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound having the structural formula described herein or an isomer thereof, wherein X is-OH; and Y is an amino acid selected from the group consisting of: histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine. In some embodiments, Y is-OH; and X is an amino acid selected from the group consisting of: histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine. In some embodiments, X and Y are each an amino acid independently selected from the group consisting of: histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine.

In some embodiments, each amino acid of the pyrrole-amino acid adducts in the methods described herein is attached through the amino group on the α -carbon of the amino acid and/or through the side chain of the amino acid. In some embodiments, each amino acid of the pyrrole-amino acid adduct is independently selected from formula IIa, formula IIb, and formula IIc:

or an isomer thereof, wherein:

each Z is independently selected from-H and-C (=O) CH ₃ The method comprises the steps of carrying out a first treatment on the surface of the And

each R is a side chain of an amino acid independently selected from the group consisting of histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, and tyrosine.

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound having a structural formula selected from the group consisting of:

and combinations thereof.

And combinations thereof.

In some embodiments, each amino acid of the pyrrole-amino acid adduct is independently selected from formula IIIa and formula IIIb:

or an isomer thereof, wherein:

each Z is independently selected from-H and-C (=O) CH ₃ ；

Each R ¹ A side chain of an amino acid independently selected from the group consisting of histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, serine, threonine, tryptophan, and tyrosine; and

each R ² Is a side chain of an amino acid independently selected from histidine, cysteine, lysine, methionine, arginine, asparagine, glutamine and tryptophan.

In some embodiments, each Z of formula IIIb is-H; and the pyrrole-amino acid adduct is a compound having a structural formula selected from the group consisting of:

and combinations thereof.

In some embodiments, at least one Z of formula IIIb is-C (=o) CH ₃ The method comprises the steps of carrying out a first treatment on the surface of the And the pyrrole-amino acid adduct is a compound having a structural formula selected from the group consisting of:

and combinations thereof.

In some embodiments, when each amino acid of the pyrrole-amino acid adduct is independently selected from formula IIIa and formula IIIb, each R ¹ A side chain of an amino acid independently selected from histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, glutamine and tryptophan; each R ² Is a side chain of an amino acid independently selected from histidine, cysteine, lysine, methionine and arginine.

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound according to formula IV:

or an isomer thereof, wherein:

y is selected from:

wherein:

z is independently selected from-H and-C (=O) CH ₃ ；

R ¹ A side chain of an amino acid independently selected from histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, glutamine and tryptophan; and

R ² is a side chain of an amino acid independently selected from histidine, cysteine, lysine, methionine and arginine.

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound according to formula V:

or an isomer thereof, wherein:

X is selected from:

wherein:

z is independently selected from-H and-C (=O) CH ₃ ；

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound according to formula VI:

or an isomer thereof, wherein:

x and Y are independently selected from:

wherein:

each Z is independently selected from-H and-C (=O) CH ₃ ；

Each R ¹ A side chain of an amino acid independently selected from histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, glutamine and tryptophan; and

each R ² Is independently selected fromSide chains of amino acids histidine, cysteine, lysine, methionine and arginine.

In some embodiments, when each amino acid of the pyrrole-amino acid adduct is independently selected from formula IIIa and formula IIIb, each R ¹ And R is ² Is a side chain of an amino acid independently selected from histidine, cysteine and lysine.

In some embodiments, the pyrrole-amino acid adducts in the methods described herein are compounds selected from the group consisting of:

and combinations thereof.

In some embodiments, the pyrrole-amino acid adduct in the methods described herein is a compound having the structure:

in some embodiments, the concentration or amount of the pyrrole-amino acid adduct in the detection methods described herein is the concentration of the pyrrole-amino acid adduct measured in the biological sample. In some embodiments, step (i) comprises a combined separation-detection assay. In some embodiments, the combined separation-detection assay is selected from the group consisting of LC-MS, LC-MS/MS, CE-MS, and GC-MS. In some embodiments, step (i) comprises quantifying the concentration or amount of the pyrrole-amino acid adduct measured in the biological sample using a standard calibration curve. In some embodiments, the reference control is the concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure. In some embodiments, when pyrrolizidine alkaloid exposure is detected in the target subject, the method further comprises repeating step (i) at a later time using a biological sample obtained from the target subject.

In another aspect, the invention provides a kit for detecting pyrrolizidine alkaloid exposure in a subject of interest. The kit comprises the following components: (i) Providing a reference control of an average amount of pyrrole-amino acid adduct; and (ii) instructions for use. In some embodiments, the kit further comprises one or more reagents.

In another aspect, the invention provides a method for diagnosing or monitoring pyrrolizidine alkaloid exposure in a subject of interest. The method comprises the following steps: (i) Quantitatively determining the concentration or amount of pyrrole-amino acid adducts in a biological sample obtained from the target subject; and (ii) comparing the concentration or amount of the pyrrole-amino acid adduct to a concentration or amount of a reference control, wherein an increase in the concentration or amount of the pyrrole-amino acid adduct from step (i) compared to a reference control is indicative of the presence of pyrrolizidine alkaloid exposure in the subject of interest.

In some embodiments, the concentration or amount of the pyrrole-amino acid adduct in a diagnostic or monitoring method described herein is the concentration of the pyrrole-amino acid adduct measured in the biological sample. In some embodiments, step (i) comprises a combined separation-detection assay. In some embodiments, the combined separation-detection assay is selected from the group consisting of LC-MS, LC-MS/MS, CE-MS, and GC-MS. In some embodiments, step (i) comprises quantifying the concentration or amount of the pyrrole-amino acid adduct measured in the biological sample using a standard calibration curve. In some embodiments, the reference control is the concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure. In some embodiments, the diagnostic or monitoring method further comprises repeating step (i) at a later time using the same type of biological sample from the target subject, wherein an increase in the concentration or amount of the pyrrole-amino acid adduct at a later time as compared to the original step (i) is indicative of worsening of the pyrrolizidine alkaloid exposure, and a decrease is indicative of improvement of the pyrrolizidine alkaloid exposure.

In another aspect, the invention provides methods for diagnosing and treating pyrrolizidine alkaloid exposure in a subject of interest. The method comprises the following steps: (i) Quantitatively determining the concentration or amount of pyrrole-amino acid adducts in a biological sample obtained from the target subject; (ii) Comparing the concentration or amount of the pyrrole-amino acid adduct to the concentration or amount of a reference control; (iii) Diagnosing that the target subject has pyrrolizidine alkaloid exposure when the concentration or amount of the pyrrole-amino acid adduct from step (i) is increased compared to the reference control; and (iv) administering a therapeutically effective treatment to the target subject having pyrrolizidine alkaloid exposure.

In some embodiments, the concentration or amount of the pyrrole-amino acid adduct in the methods described herein for diagnosing and treating pyrrolizidine alkaloid exposure is the concentration of the pyrrole-amino acid adduct measured in a biological sample. In some embodiments, step (i) comprises a combined separation-detection assay. In some embodiments, the combined separation-detection assay is selected from the group consisting of LC-MS, LC-MS/MS, CE-MS, and GC-MS. In some embodiments, step (i) comprises quantifying the concentration or amount of the pyrrole-amino acid adduct measured in the biological sample using a standard calibration curve. In some embodiments, the reference control is the concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure. In some embodiments, the therapeutically effective treatment for pyrrolizidine alkaloid exposure comprises one or more of the following: termination of pyrrolizidine alkaloid exposure, symptomatic treatment, diuretic therapy, laparoscopy, microcirculatory, glucocorticoid therapy, anticoagulation therapy, transjugular intrahepatic portal venous shunt (transjugular intrahepatic portosystemic shunt) or liver transplantation.

In another aspect, the invention provides methods for diagnosing and treating a disease or disorder associated with pyrrolizidine alkaloid exposure in a subject of interest. The method comprises the following steps: (i) Quantitatively determining the concentration or amount of pyrrole-amino acid adducts in a biological sample obtained from the target subject; (ii) Comparing the concentration or amount of the pyrrole-amino acid adduct to the concentration or amount of a reference control; (iii) Diagnosing that the subject has a disease or disorder associated with pyrrolizidine alkaloid exposure when the concentration or amount of the pyrrole-amino acid adduct from step (i) is increased compared to the reference control; and (iv) administering a therapeutically effective treatment to the subject suffering from a disease or condition associated with pyrrolizidine alkaloid exposure.

In some embodiments, the concentration or amount of the pyrrole-amino acid adduct in the methods described herein for diagnosing and treating a disease or disorder associated with pyrrolizidine alkaloid exposure is the concentration of the pyrrole-amino acid adduct measured in a biological sample. In some embodiments, step (i) comprises a combined separation-detection assay. In some embodiments, the combined separation-detection assay is selected from the group consisting of LC-MS, LC-MS/MS, CE-MS, and GC-MS. In some embodiments, step (i) comprises quantifying the concentration or amount of the pyrrole-amino acid adduct measured in the biological sample using a standard calibration curve. In some embodiments, the reference control is the concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure. In some embodiments, the disease or disorder associated with pyrrolizidine alkaloid exposure is pyrrolizidine alkaloid-induced liver sinus occlusion syndrome. In some embodiments, the therapeutically effective treatment for a disease or disorder associated with pyrrolizidine alkaloid exposure comprises one or more of the following: termination of pyrrolizidine alkaloid exposure, symptomatic treatment, diuretic therapy, laparoscopy, microcirculatory, glucocorticoid therapy, anticoagulant therapy, transjugular intrahepatic portal venous shunt or liver transplantation.

These and other embodiments, aspects, and objects will become more apparent when read in conjunction with the following detailed description and the accompanying drawings.

Drawings

FIG. 1 shows the structure of the senecine bases of different types of pyrrolizidine alkaloids.

FIG. 2 shows metabolic activation pathways of the inverted senecine-type and the Orthospermine-type pyrrolizidine alkaloids leading to the formation of pyrrole-protein adducts, pyrrole-peptide adducts, pyrrole-GSH adducts and pyrrole-amino acid adducts. The first 3 adducts are further degraded to pyrrole-amino acid adducts. Eventually all pyrrole-amino acid adducts are excreted in urine. DHPA, dehydropyrrolizidine alkaloids; DHP (±) -6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (pyrrolizine).

Based on the numbering convention for the pyrrole core structure shown in fig. 2, fig. 3 shows the chemical structure of the following pyrrole-amino acid adducts: pyrrole-7-cysteine; pyrrole-9-cysteine; pyrrole-9-histidine; and pyrrole-7-acetylcysteine, as shown.

FIG. 4 shows urine (A) from healthy volunteers spiked with pyrrole-7-cysteine standard; urine from healthy volunteers (B); and representative MRM chromatograms (MRM ion transitions m/z 239- > 120) of pyrrole-7-cysteines measured in urine (C) from patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure at admission.

Fig. 5 shows the measured concentration of pyrrole-7-cysteine in urine of patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure. Measurements were made at the time of admission (n=5), 1 month after admission (n=3), 2 months after admission (n=2), and 10 months after admission (n=1).

FIG. 6 shows urine (A) from healthy volunteers spiked with pyrrole-9-cysteine standard; urine from healthy volunteers (B); and representative MRM chromatograms (MRM ion transitions m/z 239- > 120) of pyrrole-9-cysteines measured in urine (C) from patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure at admission.

Fig. 7 shows measured pyrrole-9-cysteine concentrations in urine of patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure. Measurements were made at the time of admission (n=5), 2 weeks after admission (n=3), 1 month after admission (n=3) and 2 months after admission (n=2).

FIG. 8 shows urine (A) from healthy volunteers spiked with a pyrrole-9-histidine standard; urine from healthy volunteers (B); and representative MRM chromatograms (m/z 291→136 MRM ion transitions) of pyrrole-9-histidines measured in urine (C) from patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure at the time of admission.

Fig. 9 shows measured pyrrole-9-histidine concentrations in urine of patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure. Measurements were made at the time of admission (n=5), 2 weeks after admission (n=3), 1 month after admission (n=3) and 5 months after admission (n=1).

FIG. 10 shows urine (A) from healthy volunteers spiked with pyrrole-7-acetylcysteine standard; urine from healthy volunteers (B); and representative MRM chromatograms (MRM ion transitions m/z 281→118) of pyrrole-7-acetylcysteine determined in urine (C) from patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure at the time of admission.

Fig. 11 shows measured pyrrole-7-acetylcysteine concentrations in urine of patients with liver sinus occlusion syndrome induced by pyrrolizidine alkaloid exposure. Measurements were made at the time of admission (n=5), 1 week after admission (n=4), 2 weeks after admission (n=3), and 1 month after admission (n=1).

Detailed description of the invention

The present invention provides methods and kits for determining the occurrence of toxicity in a mammal caused by Pyrrolizidine Alkaloid (PA) exposure and for clinical diagnosis. In particular, the present invention relates to biomarkers for detecting PA-induced hepatotoxicity in a biological sample (e.g., urine) from a target subject, as well as non-invasive methods and kits for diagnosing or monitoring PA exposure in a target subject.

Pyrrolizidine alkaloids are esters (e.g., monoesters, diesters, or macrocyclic diesters) formed from a qianlizidine (e.g., an amino alcohol) and one or two qianlizides (e.g., aliphatic mono-or dicarboxylic acids). PA is generally divided into three groups based on the senecio-alkali structure: cepharanthine (including its 7-alpha enantiomer, heliotropine), osclerine and latifoline (fig. 1). cepharanthine-PA and osclerine-PA have unsaturated groundsel subbase and are highly hepatotoxic and genotoxic, whereas broadleaf groundsel subbase-PA has saturated groundsel subbase and is generally considered non-toxic. Of the two toxic types of pyrrolizidine alkaloids, cepharanthine-PA is usually present in plants both in the tertiary base form and in the N-oxide form. Pyrrolizidine alkaloid N-oxide is also hepatotoxic, but it has relatively low toxicity by being biotransformed in vivo into its corresponding tertiary base form.

Pyrrolizidine alkaloid exposure, which may also be referred to herein as pyrrolizidine alkaloid poisoning (intonation), pyrrolizidine alkaloid poisoning (poisomining) or pyrrolizidine alkaloid-related liver injury, is metabolic mediated and requires metabolic activation in the liver to exert toxicity. PA is metabolized in the liver by cytochrome P450 enzymes to dehydro-pyrrolizidine alkaloids (DHPA), which are highly unstable and readily react with nearby in vivo substances to form a range of secondary pyrrole metabolites and pyrrole adducts, for example: (i) Reaction with water to produce the hydrolysis product (±) -6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP); (ii) Interact with cellular proteins to form pyrrole-protein adducts; (iii) combining with a peptide to form a pyrrole-peptide adduct; (iv) Binding to Glutathione (GSH) to produce pyrrole-GSH adducts; and (v) reacting with a cellular amino acid to form a pyrrole-amino acid adduct (PAAA). The pyrrole adducts described above can also be produced by reaction with the hydrolysis product DHP (fig. 2). All of these pyrrole adducts have been reported to potentially cause PA-induced hepatotoxicity in vitro and/or in vivo. On the other hand, the above pyrrole adducts (ii, iii and iv) undergo further degradation to produce PAAA, plus PAAA formed via pathway (v), all of which are eventually excreted from urine (fig. 2).

Fig. 3 shows the chemical structures of four PAAA representative of a series of PAAA, which are mainly derived from pyrrole-protein, pyrrole-peptide, pyrrole-GSH and/or pyrrole-amino acid adducts formed by covalent attachment of proteins, peptides, GSH or amino acids to reactive dehydro-pyrrolizidine alkaloids (DHPA). Thus, PAAA can be used as a specific biomarker of PA exposure in mammals as a metabolite of pyrrolizidine alkaloids. According to the invention, a combined chromatography-mass spectrometry analysis (e.g., LC-MS) is used to identify and quantify PAAA present in a urine sample obtained from a subject suspected of having PA-induced hepatotoxicity. The present invention provides methods for diagnosing and treating pyrrolizidine alkaloid exposure and diseases or conditions associated therewith using a combination of chromatographic-mass spectrometry to detect and quantify PAAA in urine samples.

I. Definition of the definition

Unless specifically stated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are defined.

The terms "a," "an," or "the" as used herein include not only elements having one member, but also elements having more than one member. For example, the singular forms "a," "an," or "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a PAAA" or "PAAA" includes a plurality of PAAA molecules, etc., unless the context clearly dictates otherwise.

As used herein, the term "or" is generally employed in its sense including "and/or" unless the context clearly dictates otherwise.

As used herein, the terms "about" and "approximately" and the like are used herein to modify a numerical value and represent a defined range around that value. If "X" is a value, then "about X" or "about X" generally means a value from 0.90X to 1.10X. Any reference to "about X" at least means the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X,1.09X, and 1.10X. Thus, "about X" is intended to disclose, for example, "0.98X". When "about" is applied to the beginning of a numerical range, it applies to both endpoints of the range. Thus, "about 6 to 8.5" corresponds to "about 6 to about 8.5". When "about" is applied to a first value of a set of values, it is applied to all values in the set. Thus, "about 7, 9, or 11%" corresponds to "about 7%, about 9%, or about 11%".

As used herein, the terms "comprise" and "comprising" are intended to mean that the compounds, compositions, methods, kits, and their respective components include the stated elements, but do not exclude other elements. "consisting essentially of … …" refers to those elements required for a given embodiment. The phrase allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of a given embodiment (e.g., a compound, composition, method, or kit). "consisting of … …" means a compound, composition, method, kit, and respective components thereof as described herein, excluding any elements not stated in the description of the embodiments. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

As used herein, the term "concentration" or "amount" refers to the amount of a biomarker of interest (i.e., PAAA) present in a sample. The amount may be expressed as an absolute value reflecting the total amount or total mass of the detectable biomarker in the sample volume; alternatively, the amount may be expressed as a relative value compared to another biomarker value (e.g., the concentration of the biomarker in the sample). For example, the amount or concentration of PAAA in a sample can be an amount or concentration greater than a control level or normal level of a biomarker typically present in the sample.

As used herein, the term "concentration" refers to the amount of a substance within each defined space. The concentration is typically expressed in terms of mass per unit volume or moles per unit volume. In the context of the present invention, the concentration or amount of PAAA detected or quantitatively determined in a biological sample (i.e. urine sample) is the concentration of PAAA molecules in the volume of urine solution. In some embodiments, PAAA concentration may be expressed in molar concentration (i.e., moles of PAAA per liter of solution), such as pM, nM, μm, mM, M, etc. In some embodiments, PAAA concentration may be expressed in terms of mass concentration (i.e., mass of PAAA per liter of solution), e.g., pg/mL, mg/dL, etc.

As used herein, the term "biological sample" or "sample" refers to a plurality of sample types obtained or isolated from a subject, which can be used in any of the methods described herein. The biological sample may comprise any biological material suitable for detecting a desired biomarker (i.e., the pyrrole-amino acid adducts described herein), and may comprise cellular and/or non-cellular material from a subject. The sample may be isolated from any suitable biological tissue or fluid, such as saliva, blood, plasma, serum, urine or liver tissue. If desired, the sample may be pre-treated, for example, for enrichment or separation.

As used herein, the term "biomarker" refers to a pyrrolizidine alkaloid-derived pyrrole-amino acid adduct (i.e., a pyrrole-amino acid adduct, or PAAA, described in further detail below). In the context of the present invention, the term "pyrrole moiety" refers to the fused ring system of the pyrrole-amino acid adducts described in detail herein, which has the general 1, 7-substituted-2, 3-dihydro-1H-pyrrolizine chemical structure as shown below:

as used herein, the term "amino acid" refers to naturally occurring alpha amino carboxylic acids, as well as optical isomers (enantiomers and diastereomers), synthetic amino acid analogs, amino acid mimics that function in a manner similar to naturally occurring amino acids, and derivatives thereof. The alpha-amino acid comprises a carbon atom (i.e., an alpha-carbon) bonded to a carboxyl group, an amino group, a hydrogen atom, and a unique "side chain" group. Naturally occurring amino acids are those encoded by the genetic code and include those amino acids which are later modified (e.g., hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine), and include the L-isomers of glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine. For the purposes of this application, a synthetic amino acid analog refers to a compound having the same basic chemical structure as a naturally occurring amino acid (i.e., carbon bonded to hydrogen, carboxyl, amino, and R groups), e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. For the purposes of this application, amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids may include those having non-naturally occurring D-chirality as disclosed in WO01/12654, which may improve the stability (e.g. half life), bioavailability and other properties of the amino acid. Amino acids may be represented herein by commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee.

As used herein, the term "amino acid side chain" or "side chain" refers to a group or moiety attached to the alpha-carbon of an amino acid. For example, the alanine side chain is a fingernail (i.e., -CH ₃ ) Lysine side chain refers to a butylene-amine group (i.e., - (CH) ₂ ) ₄ NH ₂ ) And the cysteine side chain refers to a methylene-thiol group (i.e., -CH ₂ SH)。

As used herein, the term "isomer" refers to a compound (i.e., PAAA compound, biomarker, or metabolite as described herein) that has the same molecular formula and molecular weight as another compound, but differs in one or more physical and/or chemical properties. Such isomers have the same number and type of atoms, but differ in the relative positions or spatial arrangement of the constituent atoms in the molecule. There are two main isomeric forms: structural isomers (e.g., geometric isomers, tautomers, etc.) and spatial isomers (e.g., stereoisomers, enantiomers, diastereomers, etc.). Thus, the term "isomer" refers to PAAA compounds having asymmetric carbon atoms (optical centers) or double bonds, and racemates, diastereomers, enantiomers, geometric isomers, structural isomers and individual isomers thereof are intended to be encompassed within the scope of the present invention.

As used herein, the term "tautomer" refers to a structural isomer of an organic compound (e.g., PAAA biomarker) that is readily transformed by chemical reaction of tautomerism or tautomerism. This reaction generally results in the formal migration of hydrogen atoms or protons, accompanied by a transition between single bonds and adjacent double bonds. Tautomerism is a special case of structural isomerism, and because of rapid tautomerism, tautomers are generally considered to be the same compounds. In solutions where tautomerization may occur, chemical equilibrium of the tautomer will be reached. The exact ratio of tautomers depends on several factors including, but not limited to, temperature, solvent and pH. Exemplary common tautomeric pairs include, but are not limited to, keto and enol, enamine and imine, ketene and alkynol, nitroso and oxime, amide and imidic acid, lactam and lactam (amide and imidic acid tautomerism in heterocycles), enamine and enamine, and anomer of reducing sugars.

As used herein, the term "stereoisomer" refers to a heterogeneous molecule (e.g., PAAA biomarker) that has the same molecular formula and sequence of bonded atoms (i.e., make-up), but whose atoms differ in their three-dimensional orientation in space. This is in contrast to structural isomers, which share the same molecular formula but differ in the linkage or sequence thereof. Molecules that are stereoisomers from each other, by definition, represent the same structural isomer. Enantiomers are two stereoisomers that are related by reflection and are non-superimposable mirror images. Each stereocenter in one enantiomer has the opposite configuration in the other enantiomer. Two compounds that are enantiomers to each other have the same physical properties, except for the direction in which they rotate polarized light and the manner in which they interact with different optical isomers of the other compounds. Diastereomers and stereoisomers which are not related by reflection manipulation are not mirror images of each other. These include meso compounds, cis-and trans- (E-and Z-) isomers, and diastereoisomeric optical isomers. Diastereoisomers rarely have the same physical properties. For purposes of this disclosure, stereoisomers may refer to enantiomers, diastereomers, or both. Stereoisomers are generally obtained in partially purified form. For compounds of the invention having stereoisomers, such partially purified forms include those having 60%, 70%, 80%, 90% or 95% of one major stereoisomer.

As used herein, the term "positional isomer" or "structural isomer" refers to different compounds (e.g., PAAA biomarkers) that have the same number and type of atoms and thus the same molecular weight but with different atom linkages.

As used herein, the term "stereochemically pure" in reference to a compound (e.g., PAAA biomarker) as used herein refers to a compound or a composition thereof that comprises predominantly one stereoisomer of the compound and is substantially free of other stereoisomers of the compound. For example, a stereochemically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereochemically pure composition of a compound having two or more chiral centers will be substantially free of other diastereomers of the compound. Typical stereochemically pure compounds contain about 80% by weight or more of one stereoisomer of the compound and about 20% by weight or less of the other stereoisomers of the compound. For example, in various embodiments, a stereochemically pure compound comprises 90% by weight or more of one stereoisomer of the compound and about 10% by weight or less of the other stereoisomer of the compound; about 95% by weight or more of one stereoisomer of the compound and about 5% by weight or less of the other stereoisomers of the compound; about 97% by weight or more of one stereoisomer of the compound and about 3% by weight or less of the other stereoisomers of the compound; about 98% by weight or more of one stereoisomer of the compound and about 2% by weight or less of the other stereoisomers of the compound and about 99% by weight or more of one stereoisomer of the compound and about 1% by weight or less of the other stereoisomers of the compound.

As used herein, the term "derivative" refers to a compound obtained by substituting a part of the structure of a compound with another group or an atomic group.

As used herein, the term "reference control" refers to a predetermined amount or concentration of PAAA that serves as a basis for comparing the amount of PAAA present in a biological sample. The reference control may be based on a single sample, e.g., an amount or concentration obtained from a sample obtained at an earlier point in time from the same target subject to be tested, or an amount or concentration obtained from a sample from a patient exposed to other PA than the individual being tested, or a normal subject, which is a subject with no PA exposure or no PA exposure (i.e., a healthy subject). The reference control may be based on a bulk sample, e.g. from a PA-exposed patient or normal individual, or on a sample cell comprising the sample to be tested. The amount or concentration of such reference control may also be adjusted according to the specific technique (e.g., LC-MS, GC-MS, etc.) used to measure the concentration of the biomarker in the biological sample, which may vary based on the specific technique used. The reference control may be an absolute value, a relative value, a value having an upper or lower limit, a range of values, an average, a median, a mean, or a value compared to a particular control or baseline value. In some embodiments, the reference control is a concentration or amount of PAAA measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure, wherein the reference control amount or concentration is undetectable or zero nM. In other words, when the reference control is based on a biological sample obtained from a healthy subject, the amount or concentration of PAAA detected in the sample is zero nM or the amount or concentration of PAAA in the sample is undetectable. Thus, the amount or concentration of PAAA contained in a urine sample obtained from a healthy subject (i.e., a subject without PA exposure) is zero nM (i.e., 0 nM), or undetectable.

As used herein, "increase" or "decrease" refers to a detectable positive or negative change from the amount of a comparison control (i.e., an established reference control), such as the average amount of pyrrole-amino acid adducts found in a biological sample obtained from a subject not having PA exposure (i.e., a PAAA-free urine sample). The increase is a positive change, which is typically at least 10%, or at least 20%, or 50%, or 100% of the reference control, and can be as high as at least 2-fold or at least 5-fold or even 10-fold of the reference control. Similarly, the decrease is a negative change, which is typically at least 10%, or at least 20%, 30% or 50%, or even up to at least 80% or 90% of the control value. Other terms are used in this application in the same manner as described above to refer to variations or differences in quantity based on comparison, such as "more," less, "" higher, "and" lower. Conversely, the term "substantially the same" or "substantially no change" means that there is little to no change in quantity from the reference control value, typically within ±10% of the reference control, or within ±5%, ±2% of the reference control, or even less variation.

As used herein, the term "average" in the context of describing healthy subjects or subjects without PA exposure refers to certain characteristics (in particular the amount of PAAA found in a biological sample (i.e., urine sample) of the subject) that are representative of a randomly selected group of healthy subjects without PA exposure and/or without a disease or disorder associated with pyrrolizidine alkaloid exposure. The selected group includes a sufficient number of subjects such that the average amount of pyrrole-amino acid adducts in a biological sample (i.e., urine sample) in these individuals reflects the corresponding amount of PAAA in the general healthy population with reasonable accuracy. Furthermore, the selected group of people typically has an age similar to the target subject whose sample is tested for PA exposure. In addition, other factors such as gender, race, medical history are also considered, and in some embodiments, a match is made between the profile of the test subject and the selected group of individuals who established the "average" value.

As used herein, the phrase "combined separation-detection analysis" refers to an online combination of one or more separation techniques and one or more spectroscopic detection techniques. Exemplary separation techniques include, but are not limited to, liquid Chromatography (LC), such as High Performance Liquid Chromatography (HPLC), gas Chromatography (GC), gel Electrophoresis (GE), or Capillary Electrophoresis (CE). Exemplary spectroscopic detection techniques include, but are not limited to, fourier Transform Infrared (FTIR), photodiode arrays (PDA), ultraviolet-visible absorption or fluorescence emission, mass Spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR). Linking one or more separation techniques with one or more spectroscopic detection techniques can result in a variety of combined analysis techniques, such as CE-MS, GC-MS, LC-MS, LC-MS/MS, LC-NMR, and the like.

As used herein, the term "on-line" or "inline" as used, for example, in "on-line automated fashion" or "on-line extraction" refers to a procedure that is performed without operator intervention. In contrast, the term "offline" as used herein refers to a procedure that requires manual intervention by an operator. Thus, if the sample is centrifuged/filtered and then the supernatant is manually loaded into the autosampler, the centrifugation/filtration and loading steps are off-line with the subsequent steps. In various embodiments of the method, one or more steps may be performed in an on-line automated manner.

As used herein, the terms "isolation", "purification" or derivatives thereof do not necessarily refer to the removal of all but the PAAA biomarker of interest from a sample. Conversely, in some embodiments, the term "isolate" or "purify" refers to a procedure that enriches the amount of one or more PAAA biomarkers of interest relative to one or more other components present in a sample. In some embodiments, the "isolation" or "purification" procedure may be used to remove one or more components in the sample that may interfere with the detection of the biomarker, e.g., one or more components that may interfere with the detection of the biomarker by mass spectrometry.

As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components due to different distributions of chemical entities as they flow around or over a fixed liquid or solid phase.

The term "liquid chromatography" or "LC" as used herein refers to a process of selective retardation of one or more components of a fluid solution as the fluid uniformly permeates through a column of finely divided material or through capillary channels. The retardation is due to the distribution of the components of the mixture between the one or more stationary phases and the bulk fluid (i.e., mobile phase) as the bulk fluid moves relative to the stationary phase. Examples of the "liquid chromatography" include Normal Phase Liquid Chromatography (NPLC), hydrophilic interaction liquid chromatography (HILIC), reversed Phase Liquid Chromatography (RPLC), high Performance Liquid Chromatography (HPLC), ultra high performance liquid chromatography (UPLC), ultra High Performance Liquid Chromatography (UHPLC), and Turbulent Flow Liquid Chromatography (TFLC) (sometimes referred to as high turbulent flow liquid chromatography (HTLC) or high throughput liquid chromatography). The terms HILIC and NPLC are used interchangeably and relate to chromatographic separations whereby the stationary phase is polar in nature (i.e., silica, cyano, amino and similar types of hydrophilic functionalized fillers).

As used herein, the term "high performance liquid chromatography" or "HPLC" (sometimes referred to as "high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is enhanced by forcing the mobile phase under pressure through a stationary phase (typically a densely packed column). The term "1-D high performance liquid chromatography" or "1-D HPLC" refers to conventional single column HPLC. The term "2-D high performance liquid chromatography" refers to a high performance liquid chromatography technique in which two HPLC columns are used in such a way that: the metabolite or biomarker and any additional components co-eluted simultaneously with the metabolite or biomarker are directed from the first HPLC column onto a second HPLC column having a different stationary phase. The stationary phase of the second HPLC column is selected such that the metabolite or biomarker and the co-eluted components are separated prior to introducing the metabolite or biomarker into the mass spectrometer.

As used herein, the term "ultra high performance liquid chromatography" or "UHPLC" (sometimes referred to as "ultra high pressure liquid chromatography") refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase through the stationary phase (typically a densely packed column with stationary phase comprising filler particles having an average diameter of less than 2 μm) at high pressure (e.g., above 6,000 psi). "ultra high performance liquid chromatography" or "UPLC" is a specific UHPLC technology from Waters Corporation.

As used herein, the term "gas chromatography" or "GC" refers to chromatography in which a sample is vaporized and injected into a carrier gas stream (nitrogen or helium) that moves through a column containing a stationary phase composed of a liquid or particulate solid and is separated into its constituent compounds according to their affinity for the stationary phase.

As used herein, the term "capillary electrophoresis" or "CE" refers to an automated analytical technique for separating molecules in a sample solution by applying a voltage across a buffer-filled capillary. Capillary electrophoresis is commonly used to separate ions that move at different speeds when a voltage is applied, depending on the size and charge of the ions. The ions are considered peaks as they pass through the detector, and the area of each peak is proportional to the concentration of ions in the sample, which allows the ions to be quantitatively determined.

As used herein, the term "large particle column" or "extraction column" refers to a chromatographic column containing particles having an average particle size greater than about 35 μm. As used herein, the term "about" means ± 10%.

As used herein, the term "analytical column" refers to a chromatographic column having sufficient chromatographic plates to effect separation of substances eluting from the column in a sample to allow for the determination of the presence or amount of PAAA biomarker or metabolite. Such columns are distinguished from large particle columns or extraction columns, which have the general purpose of separating or extracting retained material from unreserved material in order to obtain a purified sample for further analysis. As used herein, the term "about" means ± 10%. In some embodiments, the analytical column contains particles having a diameter of about 5 μm or less.

As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique by which compounds are identified by their mass. MS refers to a method of filtering, detecting and measuring ions based on their mass-to-charge ratio or "m/z". MS techniques generally include (1) ionizing a compound to form a charged compound; and (2) detecting the molecular weight of the charged compound and calculating the mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. "mass spectrometers" typically include an ionizer, a mass analyzer, and an ion detector. Typically, one or more molecules of interest are ionized and the ions are then introduced into a mass spectrometry instrument, where the ions follow a path in space that depends on mass ("m") and charge ("z") due to a combination of magnetic and electric fields. See, for example, U.S. patent No. 6,204,500 entitled "Mass Spectrometry From Surfaces"; U.S. patent No. 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry"; U.S. patent No. 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry"; U.S. Pat. No. 6,124,137 entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes"; wright et al Prostate Cancer and Prostatic Diseases 1999,2:264-76; and Merchant and Weinberger, electrophoresis 2000,21:1164-67.

As used herein, the term "operating in negative ion mode" refers to those mass spectrometry methods in which negative ions are generated and detected. The term "operating in positive ion mode" as used herein refers to those mass spectrometry methods in which positive ions are generated and detected.

As used herein, the term "ionization" or "ionization" refers to the process of generating a biomarker or PAAA ion having a net charge equal to one or more electronic units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

As used herein, the term "electron ionization" or "EI" refers to a method in which a biomarker or PAAA in the gas or vapor phase interacts with an electron stream. The impact of the electrons with the biomarker or PAAA produces biomarker or PAAA ions, which can then be subjected to mass spectrometry techniques.

As used herein, the term "chemical ionization" or "CI" refers to a method in which a reagent gas (e.g., ammonia gas) is subjected to electron impact and a biomarker, PAAA, or metabolite ion is formed by the interaction of the reagent gas ion with the biomarker, PAAA, or metabolite molecule.

As used herein, the term "fast atom bombardment" or "FAB" refers to a process in which a beam of energetic atoms (typically Xe or Ar) impinges on a non-volatile sample, desorbing and ionizing molecules contained in the sample. The sample is dissolved in a viscous liquid matrix such as glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether, 2-nitrophenyl octyl ether, sulfolane, diethanolamine and triethanolamine. The appropriate matrix for the compound or sample is selected empirically.

As used herein, the term "matrix-assisted laser desorption ionization" or "MALDI" refers to a method in which a non-volatile sample is exposed to laser irradiation that desorbs and ionizes biomarkers, PAAA, or metabolites in the sample by various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For MALDI, the sample is mixed with an energy absorbing matrix that facilitates desorption of the biomarker, PAAA, or metabolite molecules.

As used herein, the term "surface enhanced laser desorption ionization" or "SELDI" refers to another method in which a non-volatile sample is exposed to laser irradiation that desorbs and ionizes biomarkers, PAAA, or metabolites in the sample through various ionization pathways, including photoionization, protonation, deprotonation, and cluster decay. For SELDI, the sample typically binds to a surface that preferentially retains one or more biomarkers, PAAA, or metabolites of interest. As in MALDI, the method may also use energy absorbing materials to promote ionization.

As used herein, the term "electrospray ionization" or "ESI" refers to a method in which a solution passes along a short length of capillary (with a high positive or negative potential applied at the end of the capillary). The solution reaching the end of the tube is evaporated (atomized) into a jet or spray of very small droplets of solution in the solvent vapor. This mist of droplets flows through the evaporation chamber. As the droplet size decreases, the surface charge density increases until natural repulsion between like charges causes ions as well as neutral molecules to be released. Heated ESI is similar but includes a heat source for heating the sample in the capillary.

As used herein, the term "atmospheric pressure chemical ionization" or "APCI" refers to mass spectrometry similar to ESI, however APCI produces ions by ion-molecule reactions that occur within a plasma at atmospheric pressure. The plasma is maintained by a discharge between the spray capillary and the counter electrode. The ions are then extracted into the mass analyser, typically by using a set of pressure differential pumping skimmer stages (differentially pumped skimmer stages). Dry preheated N can be used ₂ Countercurrent flow of gas improves solvent removal. For analysis of less polar materials, gas phase ionization in APCI is more efficient than ESI.

As used herein, the term "atmospheric photo ionization" or "APPI" refers to a process in which the photoionization mechanism of a molecule M is photon absorption and electron emission to form a molecular ion M ⁺ Is a mass spectrum of (a). Because photon energy is typically just above ionization potential, molecular ions are not readily availableAnd (5) dissociation. In some embodiments, the sample is analyzed without chromatography. Molecular ions can extract H in the presence of water vapor or a protic solvent to form MH ⁺ . This tends to occur if M has a high proton affinity. Because of M ⁺ And MH ⁺ The sum is constant, so this does not affect the accuracy of the quantification. The compounds in the protic solvents are generally in the form of MH ⁺ Rather than the non-polar compounds (e.g. naphthalene or testosterone) generally forming M ⁺ . See, for example, robb et al, anal. Chem.2000,72 (15): 3653-3659.

As used herein, the term "inductively coupled plasma" or "ICP" refers to a method in which a sample interacts with a partially ionized gas at a sufficiently high temperature such that most of the elements are atomized and ionized.

As used herein, the term "field desorption" refers to a method in which a non-volatile sample is placed on an ionization surface and a strong electric field is used to generate biomarker, PAAA, or metabolite ions.

As used herein, the term "desorb" refers to removing a biomarker, PAAA, or metabolite from a surface and/or bringing a biomarker, PAAA, or metabolite into the gas phase. Laser desorption is a technique of thermally desorbing a sample containing a biomarker, PAAA or metabolite into the gas phase by laser pulses. The laser strikes the back of a specially made 96-well plate with a metal substrate. The laser pulse heats the substrate and the heat causes the sample to transfer to the gas phase. The gas phase sample is then drawn into a mass spectrometer.

As used herein, the term "selective ion monitoring" is a detection mode for a mass spectrometer in which only ions within a relatively narrow (typically about one mass unit) mass range are detected.

As used herein, a "multiplex reaction mode", sometimes referred to as "selected reaction monitoring", is a detection mode for a mass spectrometer in which precursor ions and one or more fragment ions are selectively detected.

As used herein, the terms "lower limit of quantification", "lower limit of quantification" or "LLOQ" refer to the point at which a measurement becomes quantitatively meaningful. The biomarker, PAAA, or metabolite response at this LLOQ is identifiable, discrete, and reproducible with a relative standard deviation (RSD%) of less than 20% and an accuracy of 85% to 115%.

As used herein, the term "limit of detection" or "LOD" refers to a point at which the measured value is greater than the uncertainty associated therewith. LOD is the point at which the value exceeds the uncertainty associated with its measurement and is defined as three times the RSD of the average at zero concentration.

As used herein, the terms "detect", "detection" or "detection" refer to the general behavior found or distinguished or a particular observation of a detectable PAAA compound or metabolite or biomarker.

As used herein, the term "diagnosis" or "diagnostic" refers to the identification of the nature of a medical condition of a target subject, such as PA exposure or a disease or disorder associated therewith, from the concentration or amount of PAAA detected in a biological sample obtained from the target subject. In some embodiments, the nature of the medical condition of the target subject is also diagnosed by signs and symptoms.

As used herein, the term "monitoring" refers to the act of observing, assessing and/or measuring the status of pyrrolizidine alkaloid exposure or a disease or disorder associated with PA exposure over time. Observations, evaluations, and/or measurements may be recorded in the form of one or more quantities or values. Monitoring the status of PA exposure or a disease or condition associated therewith in a subject may be performed by continuously and repeatedly measuring the concentration or amount of PAAA in a biological sample of the subject. In some embodiments of the invention, PA exposure or a disease or disorder associated therewith in a subject is monitored by repeatedly obtaining a biological sample, subjecting the sample to a combined separation-detection assay as disclosed herein, and comparing the result of the sample taken later in time with the result of the sample taken earlier in time or with a reference control to identify any change in the status of pyrrolizidine alkaloid exposure or a disease or disorder associated therewith in the subject.

As used herein, the term "status" in the context of describing PA exposure or a disease or disorder associated therewith refers to any distinguishable manifestation of PA exposure or a disease or disorder associated therewith, e.g., the concentration or amount of PAAA in a biological sample of a subject of interest. For example, the status of PA exposure or a disease or disorder associated therewith includes, but is not limited to, the presence or absence of PAAA in a biological sample; progression or alleviation of PA exposure or a disease or condition associated therewith over time; and severity of PA exposure. The term "exacerbation state" refers to the progression of PA exposure or a disease or condition associated therewith over time. The term "improved state" refers to the alleviation of PA exposure or a disease or condition associated therewith over time.

As used herein, the term "invasive" refers to a medical procedure that accesses a portion of the body. In some embodiments, entry into the body may cause the subject to feel pain during or after the procedure. For example, surgical procedures involving incisions are invasive; blood drawing or obtaining a plasma or serum sample is considered less invasive; obtaining saliva or urine samples is non-invasive.

As used herein, the term "subject" refers to an animal, such as a mammal, including but not limited to primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like.

As used herein, the term "treatment" or "treating" refers to an act that results in the elimination, reduction, alleviation, reversal or prevention or delay of the onset or recurrence of PA exposure or any symptom of a disease or disorder associated with PA exposure. Symptoms of PA exposure or diseases or conditions associated with PA exposure include abdominal distension, liver pain, ascites, anorexia, debilitation, jaundice and hepatomegaly. "treating" a condition includes administering a therapeutically effective treatment to a subject having PA exposure or suffering from a disease or disorder associated with PA exposure. Such therapeutically effective treatments include, but are not limited to, terminating PA exposure, symptomatic treatment, diuretic therapy, laparoscopy, microcirculatory therapy, glucocorticoid therapy, anticoagulation therapy, transjugular intrahepatic portal venous shunt, or liver transplantation. In some embodiments, the disease or disorder associated with PA exposure is PA-induced liver sinus occlusion syndrome. In some embodiments, PA-induced liver sinus occlusion syndrome is characterized by edema, necrosis, detachment of endothelial cells in the small hepatic sinus vein and the interlobular vein, intrahepatic congestion, portal hypertension, and liver dysfunction.

Pyrrole-amino acid adducts

The pyrrole-amino acid adducts (PAAA) described herein are compounds that have been identified as specific biomarkers of Pyrrolizidine Alkaloid (PA) exposure. PAAA is used in the methods described herein, which have been developed for definitive, non-invasive detection and clinical diagnosis of PA exposure and related diseases in mammals. Thus, described herein are a series of pyrrolizidine alkaloid derived PAAA, which are biomarkers of PA exposure as related diseases and conditions. PAAA biomarkers can be used to monitor PA exposure in urine samples. In some embodiments, the biomarker described herein is a PA metabolite.

In some embodiments, the pyrrole-amino acid adducts used in the methods described herein are compounds according to formula I:

or an isomer thereof, wherein:

In some embodiments, the pyrrole-amino acid adduct used in the methods described herein is a compound according to formula I or an isomer thereof, wherein X is-OH; and Y is an amino acid selected from the group consisting of: histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine. In some embodiments, Y is-OH; and X is an amino acid selected from the group consisting of: histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine. In some embodiments, X and Y are each an amino acid independently selected from the group consisting of: histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tryptophan and tyrosine.

In some embodiments, X and/or Y of the pyrrole-amino acid adduct according to formula I is an independently selected amino acid residue, including but not limited to: naturally occurring amino acids; an unnatural amino acid; hydrophilic amino acids; a hydrophobic amino acid; positively charged amino acids; and negatively charged amino acids. Stereoisomers of naturally occurring alpha-amino acids include, but are not limited to, D-and L-amino acids.

Naturally occurring amino acids are those encoded by the genetic code, which are in the L-configuration, and those which are subsequently modified, for example hydroxyproline, gamma-carboxyglutamic acid and O-phosphoserine. Naturally occurring α -amino acids include, but are not limited to, the following L-amino acids: alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (gin), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Suitable D-amino acids include, but are not limited to: d-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile) and D-arginine (D-Arg). D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

or an isomer thereof, wherein:

In some embodiments, the pyrrole-amino acid adducts used in the methods described herein are compounds having a structure selected from the group consisting of:

and combinations thereof, wherein:

In some embodiments, the pyrrole-amino acid adducts in the methods described herein are compounds having a structural formula selected from the group consisting of:

and combinations thereof.

and combinations thereof, wherein:

Or an isomer thereof, wherein:

each Z is independently selected from-H and-C (=O) CH ₃ ；

In some embodiments, each R of formula IIIa ¹ Independently selected from-H, -CH ₃ 、-CH(CH ₃ ) ₂ 、-CH ₂ CH(CH ₃ ) ₂ 、-CH(CH ₃ )CH ₂ CH ₃ 、-CH ₂ OH、-CH(OH)CH ₃ 、-CH ₂ SH、-CH ₂ CH ₂ SCH ₃ 、-(CH ₂ ) ₄ NH ₂ 、-(CH ₂ ) ₃ NHC(NH)NH ₂ 、-CH ₂ C(＝O)OH、-CH ₂ CH ₂ C(＝O)OH、-CH ₂ C(＝O)NH ₂ 、-CH ₂ CH ₂ C(＝O)NH ₂ 、

In some embodiments, each R of formula IIIb ² Is a side chain of an amino acid independently selected from histidine, cysteine, lysine, methionine, arginine, asparagine, glutamine and tryptophan, wherein the nitrogen-containing or sulfur-containing functional group of each side chain is suitably modified to allow the amino acid to be linked to the pyrrole moiety through the N or S atom of the side chain. Such side chain modification is carried out using common chemical coupling reactions according to methods known to those skilled in the art. Thus, in some embodiments, each R of formula IIIb ² Independently selected from:

and combinations thereof.

In some embodiments, at least one Z of formula IIIb is-C (=o) CH ₃ The method comprises the steps of carrying out a first treatment on the surface of the And the pyrrole-amino acid adduct is a chemical having a structural formula selected from the group consisting ofA compound or isomer thereof:

and combinations thereof.

In some embodiments, when each amino acid of the pyrrole-amino acid adduct is independently selected from formula IIIa and formula IIIb, each R ¹ A side chain of an amino acid independently selected from histidine, cysteine, lysine, valine, methionine, arginine, alanine, asparagine, glutamine and tryptophan; each R ² Is a side chain of an amino acid independently selected from histidine, cysteine, lysine, methionine and arginine. In other words, when each amino acid of the pyrrole-amino acid adduct is independently selected from formula IIIa and formula IIIb, each R ¹ Independently selected from-CH ₂ SH、-(CH ₂ ) ₄ NH ₂ 、-CH(CH ₃ ) ₂ 、-CH ₂ CH ₂ SCH ₃ 、-(CH ₂ ) ₃ NHC(NH)NH ₂ 、-CH ₃ 、-CH ₂ C(＝O)NH ₂ 、-CH ₂ CH ₂ C(＝O)NH ₂ And->And each R ² Independently selected from:

in some embodiments, the pyrrole-amino acid adducts used in the methods described herein are compounds according to formula IV:

Or an isomer thereof, wherein:

y is selected from:

wherein:

z is independently selected from-H and-C (=O) CH ₃ ；

In some embodiments, the pyrrole-amino acid adducts used in the methods described herein are compounds according to formula V:

or an isomer thereof, wherein:

x is selected from:

wherein:

z is independently selected from-H and-C (=O) CH ₃ ；

In some embodiments, the pyrrole-amino acid adduct used in the methods described herein is a compound according to formula VI:

or an isomer thereof, wherein:

x and Y are independently selected from:

wherein:

Each Z is independently selected from-H and-C (=O) CH ₃ ；

each R ² Is a side chain of an amino acid independently selected from histidine, cysteine, lysine, methionine and arginine.

In some embodiments, when each amino acid of the pyrrole-amino acid adducts of formula IV, formula V, and/or formula VI is independently selected from formula IIIa and formula IIIb, each R ¹ And R is ² Is a side chain of an amino acid independently selected from histidine, cysteine and lysine. In other words, when each amino acid of the pyrrole-amino acid adducts of formula IV, formula V and/or formula VI is independently selected from formula IIIa and formula IIIb, each R ¹ Independently selected from-CH ₂ SH and- (CH) ₂ ) ₄ NH ₂ The method comprises the steps of carrying out a first treatment on the surface of the And each R ² Independently selected from->

In some embodiments, the pyrrole-amino acid adducts used in the methods described herein are compounds selected from the group consisting of:

and combinations thereof.

In some embodiments, the pyrrole-amino acid adducts used in the methods described herein are compounds having the following structure or isomers thereof:

methods for detection, diagnosis or treatment

In one aspect, the invention provides a method for detecting pyrrolizidine alkaloid exposure in a target subject, the method comprising: (i) Measuring the concentration or amount of the pyrrole-amino acid adduct (i.e., the compound of formula I, formula IV, formula V and/or formula VI as described herein) in a biological sample obtained from the subject of interest; (ii) Comparing the measured concentration or amount of the pyrrole-amino acid adduct to the concentration or amount of a reference control; and (iii) deeming detection of pyrrolizidine alkaloid exposure in the subject of interest when the measured concentration or amount of pyrrole-amino acid adduct in the sample is higher than the concentration or amount of a reference control. In some embodiments, the biological sample obtained from a target subject and used in any of the methods described herein is a urine sample. In some embodiments, the concentration or amount of the pyrrole-amino acid adduct in the detection methods described herein is the concentration of pyrrole-amino acid adduct measured in the biological sample.

Suitable samples include any sample that may contain pyrrole-amino acid adducts, PAAA biomarkers or metabolites (i.e., pyrrole-7-cysteine, pyrrole-9-histidine and pyrrole-7-acetylcysteine). For example, a sample obtained during the manufacture of a synthetically produced pyrrole-amino acid adduct or a commercial standard of PAAA may be analyzed to determine the composition, yield and/or purity of the manufacture or commercial standard. In some embodiments, the sample is a biological sample obtained from any biological source (e.g., animal or human). The sample may be obtained from a human, such as a blood sample, a plasma sample, a serum sample, a hair sample, a muscle sample, a urine sample, a saliva sample, a tear sample, or other tissue sample. In a preferred embodiment, the biological sample is obtained non-invasively. In some embodiments, the biological sample is blood or urine. In some embodiments, the biological sample is urine. Such a sample may be obtained, for example, from a subject suspected of having pyrrolizidine alkaloid exposure or having a disease or condition associated with pyrrolizidine alkaloid exposure, and seeking a diagnosis, prognosis or treatment thereof.

In some embodiments, step (i) of the method for detecting pyrrolizidine alkaloid exposure in a target subject comprises performing a combined separation-detection analysis on the sample. In some embodiments, the combined separation-detection assay is selected from the group consisting of LC-MS, LC-MS/MS, CE-MS, and GC-MS. In some embodiments, the sample is isolated or purified to obtain a preparation suitable for analysis by mass spectrometry. Such separation methods include chromatography, such as liquid chromatography, and may also involve additional purification, derivatization, or acidification processes that may be performed before or after chromatography. Thus, in some embodiments, a biological sample is prepared for combined separation-detection analysis by filtering, extracting, precipitating, centrifuging, diluting, combinations thereof, and the like, the sample. Protein precipitation is one method of preparing liquid biological samples (e.g., serum or plasma) for chromatography. Such protein purification methods are well known in the art. See, e.g., polson et al, journal of Chromatography B785:263-275 (2003). The biological sample may be centrifuged to separate the liquid supernatant from the precipitated proteins. The resulting supernatant may then be applied for liquid chromatography and subsequent mass spectrometry.

As used herein, the term "derivatize" refers to reacting two molecules to form a new molecule. The derivatizing agent may include dansyl chloride, an isothiocyanate group, a dinitro-fluorophenyl group, a nitrophenoxycarbonyl group, and/or an o-phthalaldehyde group, and the like. In some embodiments, after removal of the protein by centrifugation, a portion of the deproteinized supernatant is optionally derivatized. In other embodiments, the centrifuged supernatant of the biological sample is not derivatized prior to or after performing chromatography. In some embodiments, the biological sample may be acidified prior to detection analysis (i.e., mass spectrometry). In some embodiments, the biological sample may be acidified prior to separation (i.e., chromatography). In some embodiments, the biological sample is acidified with formic acid. In some embodiments, if urine is used as the biological sample, it may be derivatized with dansyl chloride before or after chromatography. In some embodiments, the urine is not derivatized in any way before or after chromatography.

Thus, the separation or purification method may comprise chromatography, preferably liquid chromatography, such as HPLC, UPLC or UHPLC. In some embodiments, the methods of the invention are performed without subjecting the sample to any derivatization or acidification methods prior to liquid chromatography or prior to mass spectrometry. Thus, in some embodiments, biological sample (a) is centrifuged; (b) filtering the supernatant; and (c) directly loading the filtered supernatant to LC-MS, LC-MS/MS, CE-MS and GC-MS.

One skilled in the art can select LC instruments and columns suitable for isolating urine samples containing PAAA biomarkers or metabolites (i.e., pyrrole-7-cysteine, pyrrole-9-histidine, and pyrrole-7-acetylcysteine). Chromatography columns typically include a medium (i.e., packing material) to facilitate separation (i.e., fractionation) of chemical moieties. The medium may comprise tiny particles. The particles include bonding surfaces that interact with various chemical moieties to facilitate separation of the chemical moieties. For example, one suitable bonding surface is a hydrophobic bonding surface, such as an alkyl bonding surface. The alkyl-bonded surface may comprise a C-4, C-8, C-12 or C-18 bonded alkyl group, especially a C-18 bonded group. The chromatographic column comprises an inlet for receiving a sample and an outlet for discharging an effluent comprising the fractionated sample. In some embodiments, the sample is applied to the column at the inlet, eluted with a solvent or solvent mixture, and discharged at the outlet. Different solvent modes can be selected for eluting PAAA(s). For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polytype (i.e., mixed) mode. In chromatography, separation of substances is achieved by variables such as choice of eluent (also referred to as "mobile phase"), elution mode, gradient conditions, temperature, etc.

In some embodiments, the biomarker may be purified by applying the sample to the column under conditions in which the biomarker is reversibly retained by the column packing material and one or more other substances are not retained. In these embodiments, a first mobile phase condition may be used in which the biomarker is retained by the column, and once the unreserved material is washed through, a second mobile phase condition may be used subsequently to remove the retained material from the column. Alternatively, the biomarker may be purified by applying the sample to a column under mobile phase conditions in which the biomarker elutes at a different rate than the one or more other materials. Such a process may enrich the amount of one or more biomarkers relative to one or more other components of the sample.

By careful selection of valves and connector tubing, two or more columns can be connected as desired so that material passes from one column to the next without any manual steps. In some embodiments, the selection of valves and piping is controlled by a preprogrammed computer to perform the necessary steps. In some embodiments, the chromatography system is also connected to a detector system, such as an MS system, in this online fashion. Thus, an operator can place a tray of samples in the autosampler and perform the remaining operations under computer control, thereby purifying and analyzing all selected samples.

After separation or purification of a biological sample (i.e., urine) via LC (e.g., HPLC, UPLC, or UHPLC), the separated or purified sample is then subjected to detection analysis via ionization. In various embodiments, PAAA present in the sample may be ionized by any method known to those skilled in the art. Mass spectrometry is performed using a mass spectrometer that includes an ion source for ionizing a fractionated sample and producing charged molecules for further analysis. For example, ionization of the sample may be performed by Electron Ionization (EI), chemical Ionization (CI), electrospray ionization (ESI), photon ionization, atmospheric Pressure Chemical Ionization (APCI), photo ionization, atmospheric photo ionization (APPI), fast Atom Bombardment (FAB), liquid Secondary Ionization (LSI), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermal spray/plasma spray ionization, surface Enhanced Laser Desorption Ionization (SELDI), inductively Coupled Plasma (ICP), and particle beam ionization. Those skilled in the art will appreciate that the choice of ionization method may be determined based on the PAAA biomarker to be measured, the sample type, the detector type, the choice of positive and negative modes, etc.

In some embodiments, PAAA is ionized in a positive mode by electrospray ionization (ESI). In a related embodiment, the PAAA ions are in a gaseous state and the inert collision gas is argon or nitrogen. In an alternative embodiment, PAAA is ionized in a positive mode by Atmospheric Pressure Chemical Ionization (APCI). In other embodiments, PAAA is ionized in negative mode by electrospray ionization (ESI) or Atmospheric Pressure Chemical Ionization (APCI).

After the sample is ionized, the positively or negatively charged ions thus generated may be analyzed to determine the mass-to-charge ratio. Suitable analyzers for determining mass to charge ratios include quadrupole rod analyzers, ion trap analyzers, and time-of-flight analyzers. Several detection modes may be used to detect ions. For example, a selective ion monitoring mode (SIM) may be used to detect selected ions, or alternatively, a scanning mode (e.g., multiple Reaction Monitoring (MRM) or Selected Reaction Monitoring (SRM)) may be used to detect ions. In some embodiments, a quadrupole rod analyzer is used to determine the mass-to-charge ratio. For example, in a "quadrupole" or "quadrupole ion trap" instrument, ions in an oscillating radio frequency field are subjected to forces proportional to the DC potential applied between the electrodes, the amplitude of the RF signal and the mass/charge ratio. The voltages and amplitudes may be selected such that only ions with a particular mass/charge ratio move the length of the quadrupole, while all other ions are deflected. Thus, quadrupole instruments can be used as both "mass filters" and "mass detectors" for ions injected into the instrument.

In some embodiments, the resolution of MS techniques may be enhanced by employing "tandem mass spectrometry" or "MS/MS". In this technique, precursor ions (also referred to as parent ions) generated by the molecules of interest may be filtered in an MS instrument, followed by fragmentation of the precursor ions to generate one or more fragment ions (also referred to as daughter ions or product ions), which are then analyzed in a second MS process. By careful selection of precursor ions, only ions generated by PAAA biomarkers pass through the fragmentation chamber where they collide with atoms of inert gas to generate fragment ions. Because precursor ions and fragment ions are produced in a reproducible manner under a given set of ionization/fragmentation conditions, such MS/MS filtration/fragmentation techniques can be used to eliminate interfering substances from biological samples.

Mass spectrometers typically provide ion scanning to a user; that is, the relative abundance of each ion with a particular mass/charge within a given range (e.g., 100 to 1000 amu). The results of the PAAA assay, i.e., mass spectra, can be correlated to the amount of PAAA in the original sample by a variety of methods known in the art. For example, given that the sampling and analysis parameters are tightly controlled, the relative abundance of a given ion can be compared to a table that converts that relative abundance to an absolute amount of the original molecule. Alternatively, calibration standards may be run with the sample and a standard calibration curve constructed based on ions generated from those standards. Using such a standard curve, the relative abundance of a given ion can be converted into an absolute amount of the original molecule. In some embodiments, an internal standard is used to generate a standard calibration curve for calculating the amount of PAAA. Methods of generating and using such standard calibration curves are well known in the art and one of ordinary skill in the art will be able to select an appropriate internal standard. Various methods for correlating the amount of ions to the amount of the original molecule will be well known to those of ordinary skill in the art.

One or more steps of the method may be performed using an automated machine. In certain embodiments, one or more purification steps are performed on-line, or all purification and mass spectrometry steps may be performed in an on-line manner.

In some embodiments, such as MS/MS (where precursor ions are separated for further fragmentation), collision Activated Dissociation (CAD) can be used to generate fragment ions for further detection. In CAD, precursor ions gain energy by collisions with inert gases and are subsequently fragmented by a process called "unimolecular decomposition". Sufficient energy must be deposited in the precursor ions so that some bonds within the ions may be broken due to the increased vibrational energy.

In some embodiments, the PAAA is detected and/or quantified using MS/MS as follows. The sample is subjected to liquid chromatography, such as HPLC, UPLC or UHPLC, the liquid solvent stream from the chromatographic column enters the heated nebulizer interface of the MS/MS analyzer, and the solvent/biomarker mixture is converted to vapor in the heating tube of the interface. The PAAA biomarkers contained in the nebulized solvent are ionized by a corona discharge needle of the interface that applies a large voltage to the nebulized solvent/PAAA mixture. Ions, such as precursor ions, pass through the aperture of the instrument and enter the first quadrupole. Quadrupole rods 1 and 3 (Q1 and Q3) are mass filters that allow ions (i.e., "precursor" and "fragment" ions) to be selected based on their mass-to-charge ratio (m/z). Quadrupole rods 2 (Q2) are collision cells in which ions are fragmented. The first quadrupole (Q1) of the mass spectrometer selects precursor PAAA ions having a specific mass to charge ratio. Precursor PAAA ions of the correct mass/charge ratio are admitted into the collision cell (Q2), while unwanted ions of any other mass/charge ratio collide with the sides of the quadrupole rods and are eliminated. The precursor ions entering Q2 collide with neutral argon molecules and fragment. This method is known as Collision Activated Dissociation (CAD). The generated fragment ions enter the quadrupole rod 3 (Q3), in which quadrupole rod 3 the fragment ions of PAAA are selected while the other ions are eliminated.

The method may involve MS/MS performed in either negative or positive ion mode. Using standard methods well known in the art, one of ordinary skill in the art is able to identify one or more fragment ions that can be used to select a particular precursor ion of PAAA in quadrupole 3 (Q3).

If the precursor ions of PAAA include alcohol or amine groups, fragment ions are typically formed that represent dehydration or deamination, respectively, of the precursor ions. In the case of precursor ions comprising alcohol groups, such fragment ions formed by dehydration are caused by the loss of one or more water molecules in the precursor ion (i.e., wherein the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 18 for the loss of one water molecule, or about 36 for the loss of two water molecules, etc.). In the case of precursor ions comprising amine groups, such fragment ions formed by deamination are caused by the loss of one or more ammonia molecules (i.e., wherein the difference in mass-to-charge ratio between the precursor ion and the fragment ion is about 17 for the loss of one ammonia molecule, or about 34 for the loss of two ammonia molecules, etc.). Likewise, precursor ions comprising one or more alcohol and amine groups typically form fragment ions that represent the loss of one or more water molecules and/or one or more ammonia molecules (i.e., wherein the difference in mass-to-charge ratio between the precursor ions and the fragment ions is about 35 for the loss of one water molecule and one ammonia molecule). In general, the fragment ions representing dehydration or deamination of the precursor ions are not specific fragment ions of a particular biomarker. Thus, in some embodiments of the invention, the MS/MS is performed such that at least one fragment ion of PAAA is detected that is not merely representative of the loss of one or more water molecules and/or the loss of one or more ammonia molecules from the precursor ion.

When ions collide with the detector, an electronic pulse is generated that is converted into a digital signal. The acquired data is transferred to a computer which plots the count of the acquired ions against time. The resulting mass spectrum is similar to the chromatogram produced in the traditional HPLC method. The area under the peak, or the amplitude of such peak, corresponding to a particular ion is measured and correlated with the amount of PAAA biomarker. In certain embodiments, the area under the curve or the amplitude of the peak of the fragment ion and/or precursor ion is measured to determine the amount of PAAA. As described above, the relative abundance of a given ion can be converted into an absolute amount of the original PAAA using a standard calibration curve based on the peaks of one or more ions of a molecular standard.

Thus, in some embodiments, step (i) of the method for detecting pyrrolizidine alkaloid exposure in a target subject comprises quantifying the concentration or amount of PAAA measured in a biological sample using a standard calibration curve. In some embodiments, the standard calibration curve is made from a synthetic standard of PAAA corresponding to PAAA detected or measured in a biological sample (e.g., urine) obtained from a target subject. In some embodiments, the standard used to prepare the standard calibration curve is a synthetic PAAA corresponding to any of the PAAA compounds of formula I, formula IV, formula V, and/or formula VI described herein that are detected or measured in a biological sample (e.g., urine) of the target subject. In some embodiments, the standard is selected from the group consisting of pyrrole-7-cysteine, pyrrole-9-histidine, pyrrole-7-acetylcysteine, and combinations thereof.

In some embodiments, the biological sample is doped with an internal calibration standard, which may optionally be isotopically labeled. Aliquots of each sample mixture can then be injected directly onto an LC-MS or LC-MS/MS system equipped with an HPLC, UPLC or UHPLC column without further processing. The peak area of the respective PAAA product ion was measured relative to the peak area of the product ion of the internal standard. In some embodiments, the quantification is performed using a weighted linear least squares regression analysis.

In some embodiments, the reference control used in the methods described herein (e.g., methods for detecting pyrrolizidine alkaloid exposure in a subject of interest) is the concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure. In some embodiments, the reference control is a concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure, wherein the reference control amount or concentration is undetectable or zero nM (i.e., 0 nM). In some embodiments, when pyrrolizidine alkaloid exposure is detected in the target subject, the method further comprises repeating step (i) at a later time using a biological sample obtained from the target subject.

In another aspect, the present invention provides a method for diagnosing or monitoring PA exposure in a target subject, the method comprising: (i) Quantitatively determining the concentration or amount of PAAA (i.e., a compound of formula I, formula IV, formula V, and/or formula VI as described herein) in a biological sample (i.e., urine) obtained from a target subject; and (ii) comparing the concentration or amount of PAAA to the concentration or amount of a reference control, wherein an increase in the concentration or amount of PAAA from step (i) compared to the reference control is indicative of PA exposure in the target subject. In some embodiments, the concentration or amount of PAAA of a diagnostic or monitoring method described herein is the concentration of PAAA measured in a biological sample (i.e., urine). In some embodiments, step (i) comprises a combined separation-detection analysis (e.g., LC-MS/MS, CE-MS, and GC-MS).

In some embodiments, step (i) comprises quantifying the concentration or amount of PAAA measured in the biological sample using a standard calibration curve. In some embodiments, the standard calibration curve is made from a synthetic standard of PAAA corresponding to PAAA measured in a biological sample (e.g., urine) obtained from a target subject. In some embodiments, the standard used to prepare the standard calibration curve is a synthetic PAAA corresponding to any of the PAAA compounds of formula I, formula IV, formula V, and/or formula VI described herein as measured in a biological sample (e.g., urine) of the target subject. In some embodiments, the standard is selected from the group consisting of pyrrole-7-cysteine, pyrrole-9-histidine, pyrrole-7-acetylcysteine, and combinations thereof.

In some embodiments, the reference control used in the method for diagnosing or monitoring PAAA is the concentration or amount of pyrrole-amino acid adduct measured in a biological sample obtained in a subject without PA exposure. In some embodiments, the reference control is a concentration or amount of PAAA measured in a biological sample obtained from a healthy subject (i.e., a subject without PA-exposure), wherein the reference control amount or concentration is 0nM or undetectable. In some embodiments, the diagnostic or monitoring method further comprises repeating step (i) at a later time using the same type of biological sample from the target subject, wherein an increase in the concentration or amount of PAAA at the later time as compared to the original step (i) is indicative of a worsening of PA exposure and a decrease is indicative of an improvement of PA exposure.

In another aspect, the present invention provides a method for diagnosing and treating PA exposure in a subject, the method comprising: (i) Quantitatively determining the concentration or amount of PAAA (i.e., a compound of formula I, formula IV, formula V, and/or formula VI as described herein) in a biological sample (i.e., urine) obtained from the target subject; (ii) Comparing the concentration or amount of PAAA to the concentration or amount of a reference control; (iii) Diagnosing that the target subject has PA exposure when the concentration or amount of PAAA from step (i) is increased compared to a reference control; and (iv) administering a therapeutically effective treatment to the subject having PA exposure. In some embodiments, the concentration or amount of PAAA in the diagnostic and therapeutic PA exposure methods described herein is the concentration of PAAA measured in a biological sample (i.e., urine). In some embodiments, step (i) comprises a combined separation-detection analysis (e.g., LC-MS/MS, CE-MS, and GC-MS).

In some embodiments, step (i) comprises quantifying the concentration or amount of PAAA measured in the biological sample using a standard calibration curve. In some embodiments, the standard calibration curve is made from a synthetic standard of PAAA corresponding to PAAA detected or measured in a biological sample (e.g., urine) obtained from a target subject. In some embodiments, the standard used to prepare the standard calibration curve is a synthetic PAAA corresponding to any of the PAAA compounds of formula I, formula IV, formula V, and/or formula VI described herein that are detected or measured in a biological sample (e.g., urine) of the target subject. In some embodiments, the standard is selected from the group consisting of pyrrole-7-cysteine, pyrrole-9-histidine, pyrrole-7-acetylcysteine, and combinations thereof.

In some embodiments, the reference control is the concentration or amount of PAAA measured in a biological sample obtained in a subject without PA exposure. In some embodiments, the reference control is a concentration or amount of PAAA measured in a biological sample obtained from a healthy subject, wherein the reference control amount or concentration is 0nM or undetectable. In some embodiments, the therapeutically effective treatment for PA exposure comprises one or more of the following: terminating PA exposure, symptomatic treatment, diuretic therapy, laparoscopy, microcirculatory therapy, glucocorticoid therapy, anticoagulation therapy, transjugular intrahepatic portal venous bypass or liver transplantation.

In another aspect, the invention provides a method for diagnosing and treating a disease or disorder associated with PA exposure in a subject, the method comprising: (i) Quantitatively determining the concentration or amount of PAAA (i.e., a compound of formula I, formula IV, formula V, and/or formula VI as described herein) in a biological sample (i.e., urine) obtained from the target subject; and (ii) comparing the concentration or amount of PAAA to the concentration or amount of a reference control; (iii) Diagnosing that the subject has a disease or disorder associated with PA exposure when the concentration or amount of PAAA from step (i) is increased compared to the reference control; and (iv) administering a therapeutically effective treatment to the subject having a disease or disorder associated with PA exposure.

In some embodiments, the concentration or amount of PAAA in the methods of diagnosing and treating a disease or disorder associated with PA exposure described herein is the concentration of PAAA measured in a biological sample (i.e., urine). In some embodiments, step (i) comprises a combined separation-detection analysis (e.g., LC-MS/MS, CE-MS, and GC-MS). In some embodiments, step (i) comprises quantifying the concentration or amount of the pyrrole-amino acid adduct measured in the biological sample using a standard calibration curve. In some embodiments, the standard calibration curve is made from a synthetic standard of PAAA corresponding to PAAA detected or measured in a biological sample (e.g., urine) obtained from a target subject. In some embodiments, the standard used to prepare the standard calibration curve is a synthetic PAAA corresponding to any of the PAAA compounds of formula I, formula IV, formula V, and/or formula VI described herein that are detected or measured in a biological sample (e.g., urine) of the target subject. In some embodiments, the standard is selected from the group consisting of pyrrole-7-cysteine, pyrrole-9-histidine, pyrrole-7-acetylcysteine, and combinations thereof.

In some embodiments, the reference control is the concentration or amount of PAAA measured in a biological sample obtained in a subject without PA exposure. In some embodiments, the reference control is a concentration or amount of PAAA measured in a biological sample obtained from a healthy subject, wherein the reference control amount or concentration is undetectable or 0nM. In some embodiments, the disease or disorder associated with PA exposure is PA-induced liver sinus occlusion syndrome. In some embodiments, the therapeutically effective treatment for a disease or disorder associated with PA exposure comprises one or more of the following: terminating PA exposure, symptomatic treatment, diuretic therapy, laparoscopy, microcirculatory therapy, glucocorticoid therapy, anticoagulation therapy, transjugular intrahepatic portal venous bypass or liver transplantation.

IV. kit

The invention also provides kits to facilitate and/or normalize pyrrole-amino acid adducts for use in methods for detecting pyrrolizidine alkaloid exposure in a subject of interest. Materials and reagents for performing these various methods may be provided in the kit to facilitate the performance of the methods. As used herein, the term "kit" includes a combination of items that facilitate a method, assay, analysis, or operation.

In some embodiments, the kit comprises the following components: (i) Providing a reference control of an average amount of pyrrole-amino acid adduct; and (ii) instructions for use. The reference control represents the average of the pyrrole-amino acid adducts in a biological sample (e.g., urine) of a healthy subject not having pyrrolizidine alkaloid exposure. In some cases, the reference control may be provided in the form of a set point. In some embodiments, the instructions provided in the kits of the invention are instruction manuals that direct a user to detect, diagnose, or monitor pyrrolizidine alkaloid exposure in a subject of interest.

In some embodiments, the kit further comprises one or more reagents. For example, reagents, solutions, buffers or other chemical reagents for sample collection and/or purification, suitable samples for normalizing and/or normalizing the sample. In some embodiments, the kit further comprises one or more standards for preparing the pyrrole-amino acid adducts of the standard calibration curve. In some embodiments, the standard is a synthetic PAAA corresponding to PAAA detected or measured in a biological sample (e.g., urine) of the target subject. The standard calibration curve prepared from synthetic standards of PAAA contained in the kit can be used to quantify the concentration or amount of the corresponding PAAA detected in a biological sample obtained from a target subject. In some embodiments, the standard included in the kit is a synthetic PAAA corresponding to any of the PAAA compounds of formula I, formula IV, formula V, and/or formula VI described herein that are detected or measured in a biological sample (e.g., urine) of a target subject. In some embodiments, the kit comprises synthetic standards for PAAA for preparing one or more calibration curves, wherein the standards are selected from the group consisting of pyrrole-7-cysteine, pyrrole-9-histidine, pyrrole-7-acetylcysteine, and combinations thereof.

Furthermore, the kits of the present invention may include, but are not limited to, devices for sample collection and/or purification, sample tubes, racks, trays, plates, and the like. The kits of the invention may also be packaged for convenient storage and safe transport, for example in a box with a lid.

Examples

The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially the same or similar results.

A chemical. Four representative biomarkers shown in fig. 3 (i.e., pyrrole-7-cysteine, pyrrole-9-histidine and pyrrole-7-acetylcysteine) were synthesized and quantified based on well established procedures previously reported publicly. See He et al, j.food Drug al.2016,24 (4), 682-694; and He et al, J.Environ.Sci.health C2017,35 (2), 69-83. Briefly, commercially available inverted thousand-base pyrrolizidine alkaloids, monocrotaline, are reacted with o-tetrachloroquinone to produce dehydro-monocrotaline. Then, dehydro-monocrotaline is reacted with cysteine, histidine or acetylcysteine, respectively, to produce pyrrole-7-cysteine, pyrrole-9-histidine and pyrrole-7-acetylcysteine. Each synthetic biomarker was purified from their respective reaction mixtures and their structures were confirmed by MS and NMR spectra. All solvents used were HPLC grade.

Urine PAAA analysis in patients with pyrrolizidine alkaloid-induced liver injury.

In this study, urine samples were provided by patients suffering from liver injury who were admitted to the drummer hospital at the university of south Beijing, jiangsu province, china. All patients consumed pyrrolizidine alkaloid containing herbs prior to onset of liver damage. Clinical diagnosis of pyrrolizidine alkaloid induced liver injury is performed according to < < south Beijing criteria > >, including patient confirmed PA intake history, clinical symptoms/requirements: 1) Abdominal distension and/or pain in the liver area, hepatomegaly and ascites; 2) Elevated total serum bilirubin or laboratory liver examination abnormalities; 3) Evidence of heterogeneity and enhancement of liver using CT or MRI; and/or 4) exclude other pathological evidence of known causes of liver injury. See Zhuge, Y et al, j. Gastroentol. Hepatol.2019,34 (4), 634-642. The collected urine samples were stored at-80 ℃ until ready for identification and quantification of PAAA biomarkers (i.e., pyrrole 7-cysteine, pyrrole 9-histidine and pyrrole 7-acetylcysteine) using the LC-MS/MS method developed below.

An aliquot of the urine sample (200. Mu.L) was centrifuged at 21, 130g for 15min at 4 ℃. The supernatant was collected and filtered for combined chromatography-mass spectrometry performed on an Agilent 6460 triple quadrupole LC-MS system using a Waters Acquity BEH C column (2.1 x 100mm,1.7 μm). A binary LC gradient is applied to the analytical column to separate the pyrrole-amino acid adducts of interest from the urine sample. Mobile phase a was water containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid, which was applied in an LC gradient elution method as follows: 2% B at 0-0.5 min; 2-15% B at 0.5-20 min; 15-18% B at 20-22 min; 18% b at 22-23 min; 18-60% B at 23-24 min; 24-27 minutes, 60% B. The flow rate was 0.3mL/min, the column temperature was maintained at 45℃and the sample volume was 2. Mu.L. The mass spectrometer was operated using Multiple Reaction Monitoring (MRM) in positive ion mode with electrospray ionization (ESI) interface. The mass spectrometer parameters used were as follows: the gas temperature is 320 ℃; the gas flow rate is 7L/min; nebulizer gas at 45 psi; the capillary voltage was 4000V. Agilent MassHunter Workstation (B.06.00) is used for LC-MS/MS system operation, data acquisition and processing. Data analysis was performed by GraphPad Prism 6.0.

The LC-MS/MS method developed above was successfully implemented to identify and quantify the pyrrole-7-cysteine adducts present in urine samples obtained from patients with pyrrolizidine alkaloid poisoning. In the presence of precursor ions (i.e., [ M+H-H ] ₂ O] ⁺ ) To the product ion (i.e., [ M+H-H) ₂ O-C ₃ H ₅ NO ₂ S] ⁺ ) Pyrrole-7-cysteines were monitored at the m/z 239→m/z 120 ion transition. Representative MRM chromatograms of pyrrole-7-cysteine in urine (a) spiked with pyrrole-7-cysteine standard from healthy volunteers, urine (B) from healthy volunteers, and urine (C) from patients with PA exposure are shown in fig. 4. The MRM chromatogram of the blank urine from healthy volunteers (fig. 4, panel B) did not show any interfering signal at the retention time of pyrrole-7-cysteine.

Calibration curves for quantifying pyrrole-7-cysteine in urine samples were generated by analyzing serial diluted stock solutions of the synthetic standard pyrrole-7-cysteine compound at different concentrations using the LC-MS/MS method described above. Briefly, pyrrole-7-cysteine synthesis standard was dissolved in water to produce a stock solution. Stock solutions were then serially diluted with blank urine (which did not contain pyrrole-7-cysteine) obtained from healthy volunteers to prepare pyrrole-7-cysteine samples at different concentrations. LC-MS/MS analysis was then performed on each pyrrole-7-cysteine sample. Peak areas of pyrrole-7-cysteines from each MRM chromatogram were plotted against the concentration of each pyrrole-7-cysteine sample over a concentration range of 2.70 to 540.54nM to yield a regression equation of y=4.8820x+17.5259 and correlation coefficient (r ² ) A linear 7-point calibration curve of 0.994. The lower limit of quantitation (LLOQ) was determined to be 2.70nM and the limit of detection (LOD) was determined to be 0.81nM.The concentration of pyrrole-7-cysteine detected in urine samples from PA-exposed patients was determined based on a calibration curve. The results show that pyrrole-7-cysteine was detected at the time of admission (n=5), 1 month after admission (n=3), 2 months after admission (n=2) and 10 months after admission (n=1) from all urine samples collected from different PA-exposed patients. The concentration of the biomarker in urine varied from patient to patient and from sample to sample at different time points from the same patient, the highest concentration of pyrrole-7-cysteine detected was 63.62nM and the lowest concentration of pyrrole-7-cysteine detected was 4.09nM (FIG. 5).

The same LC-MS/MS method described above was used to identify and quantify pyrrole-9-cysteine adducts present in urine samples obtained from patients with PA poisoning (i.e., PA exposure). In the presence of precursor ions (i.e. [ M+H-H ] ₂ O] ⁺ ) To the product ion (i.e. [ M+H-H ] ₂ O-C ₃ H ₅ NO ₂ S] ⁺ ) Pyrrole-9-cysteines were monitored at the m/z 239→m/z 120 ion transition. Representative MRM chromatograms of pyrrole-9-cysteine in urine (a) spiked with pyrrole-9-cysteine standard from healthy volunteers, urine (B) from healthy volunteers, and urine (C) from patients with PA exposure are shown in fig. 6. The MRM chromatogram of the blank urine from healthy volunteers (fig. 6, panel B) did not show any interfering signal at the retention time of pyrrole-9-cysteine.

Calibration curves for quantifying pyrrole-9-cysteine in urine samples were generated by analyzing serial diluted stock solutions of the synthetic standard pyrrole-9-cysteine compound at different concentrations using the LC-MS/MS method described above. Briefly, pyrrole-9-cysteine synthesis standards were dissolved in water to produce stock solutions, which were then serially diluted with blank urine (i.e., 0nM pyrrole-9-cysteine) obtained from healthy volunteers to prepare samples of different pyrrole-9-cysteine concentrations. LC-MS/MS analysis was then performed on each pyrrole-9-cysteine sample. Peak areas of pyrrole-9-cysteines from each MRM chromatogram were plotted against the concentration of each pyrrole-9-cysteine sample over a concentration range of 2.70 to 540.54nMTo yield a regression equation of y=7.6398x+22.1566 and a correlation coefficient (r ² ) A linear 7-point calibration curve of 0.996. The lower limit of quantitation (LLOQ) was determined to be 2.70nM and the limit of detection (LOD) was determined to be 0.81nM. The concentration of pyrrole-9-cysteine detected in urine samples from PA-exposed patients was determined based on a calibration curve. The results show that pyrrole-9-cysteine was detected at the time of admission (n=5), 2 weeks after admission (n=3), 1 month after admission (n=3) and 2 months after admission (n=2) from all urine samples taken from different PA-exposed patients. The highest concentration of pyrrole-9-cysteine detected in the urine of the patient was 35.63nM, while the lowest concentration detected was 2.32nM (FIG. 7).

Likewise, the same LC-MS/MS method described above was used to identify and quantify pyrrole-9-histidine adducts in urine of patients with PA poisoning. In the presence of precursor ions (i.e., [ M+H ]] ⁺ ) To the product ion (i.e., [ M+H-C) ₆ H ₉ N ₃ O ₂ ] ⁺ ) Pyrrole-9-histidines were monitored at the m/z 291. Fwdarw.m/z 136 ion transition. Representative MRM chromatograms of pyrrole-9-histidine in urine (a) spiked with pyrrole-9-histidine standard from healthy volunteers, urine (B) from healthy volunteers, and urine (C) from patients with PA exposure are shown in fig. 8. The MRM chromatogram of the blank urine from healthy volunteers (fig. 8, panel B) did not show any interfering signal at the retention time of pyrrole-9-histidine.

As described above, calibration curves for quantifying pyrrole-9-histidine in urine samples were generated by serial dilution of stock solutions of the synthetic standard pyrrole-9-histidine compound at different concentrations. LC-MS/MS analysis was performed on each pyrrole-9-histidine standard sample, and the peak area of pyrrole-9-histidine in each MRM chromatogram was plotted against the pyrrole-9-histidine concentration in each sample, resulting in a linear 7-point calibration curve with a concentration range of 0.54 to 270.27nM, having a linear regression equation of y=12.3777x+3.3380 and a correlation coefficient (r ² ) 0.994. The lower limit of quantitation (LLOQ) was determined to be 0.54nM and the limit of detection (LOD) was determined to be 0.16nM. Determination of PA exposed patients based on calibration curvesThe concentration of pyrrole-9-histidine detected in the urine sample. The results show that pyrrole-9-histidine was detected at the time of admission (n=5), 2 weeks after admission (n=3), 1 month after admission (n=3) and 5 months after admission (n=1) from all urine samples collected from different PA-exposed patients. The highest concentration of pyrrole-9-histidine detected in the urine of the patient was 32.61nM, while the lowest concentration detected was 6.95nM (FIG. 9).

Identification and quantification of pyrrole-7-acetylcysteine adducts in urine obtained from PA-exposed patients was performed according to the LC-MS/MS method described herein. In the presence of precursor ions (i.e. [ M+H-H ] ₂ O] ⁺ ) To the product ion (i.e. [ M+H-H ] ₂ O-C ₅ H ₉ NO ₃ S] ⁺ ) Is monitored for pyrrole-7-acetylcysteine at the m/z281→mz 118 ion transition. Representative MRM chromatograms of pyrrole-7-acetylcysteine in urine (a) spiked with pyrrole-7-acetylcysteine standard from healthy volunteers, urine (B) from healthy volunteers, and urine (C) from patients with PA exposure are shown in fig. 10. The MRM chromatogram of the blank urine from healthy volunteers (fig. 10, panel B) did not show any interfering signal at the retention time of pyrrole-7-acetylcysteine.

As described above, calibration curves for quantifying pyrrole-7-acetylcysteine in urine samples were generated by serial dilution of stock solutions of the synthetic standard pyrrole-7-acetylcysteine compound at different concentrations. LC-MS/MS analysis was performed on each pyrrole-7-acetylcysteine standard sample, and the peak area of pyrrole-7-acetylcysteine in each MRM chromatogram was plotted against the pyrrole-7-acetylcysteine concentration in each sample, thereby generating a linear 7-point calibration curve with a concentration range of 9.43 to 943.24nM, the linear regression equation of the calibration curve for pyrrole-7-acetylcysteine being y= 13.1397x-6.4272 and the correlation coefficient (r ² ) 0.997. The lower limit of quantitation (LLOQ) was determined to be 9.43nM and the limit of detection (LOD) was determined to be 2.82nM. The concentration of pyrrole-7-acetylcysteine detected in urine samples of PA-exposed patients was determined based on a calibration curve. The results showed that at the time of admission (n=5), after admissionPyrrole-7-acetylcysteine was detected in all urine samples collected from different PA-exposed patients for 1 week (n=4), 2 weeks after admission (n=3), and 1 month after admission (n=1). The highest concentration of pyrrole-7-acetylcysteine detected in the urine of the patient was 34.78nM, while the lowest concentration detected was 11.08nM (FIG. 11).

In summary, four representative PAAAs were detected in all urine samples taken from patients with pyrrolizidine alkaloid induced liver injury, whereas none of these four representative PAAAs were detected in urine of healthy volunteers. The results demonstrate that a single representative PAAA and/or several PAAAs in combination can be used as specific non-invasive biomarkers of pyrrolizidine alkaloid exposure in humans and can be used for clinical diagnosis of pyrrolizidine alkaloid-induced liver injury.

Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood by those of ordinary skill in the art that certain changes and modifications may be practiced within the scope of the appended claims.

Reference to the literature

Xiaobo He,Liang Ma,Qingsu Xia,and Peter P.Fu.7-N-Acetylcysteine-pyrrole conjugate-A potent DNA reactive metabolite of pyrrolizidine alkaloids.Journal of Food and Drug Analysis 24,no.4(2016):682-694.

Xiaobo He,Qingsu Xia,and Peter P.Fu.7-Glutathione-pyrrole and 7-cysteine-pyrrole are potential carcinogenic metabolites of pyrrolizidine alkaloids.Journal of Environmental Science and Health,Part C 35,no.2(2017):69-83.

Yuzheng Zhuge,Yulan Liu,Weifen Xie,Xiaoping Zou,Jianming Xu,Jiyao Wang and Chinese Society of Gastroenterology Committee of Hepatobiliary Disease.Expert consensus on the clinical management of pyrrolizidine alkaloid-induced hepatic sinusoidal obstruction syndrome.Journal of Gastroenterology and Hepatology 34,no.4(2019):634-642.

Claims

1. Use of an pyrrole-amino acid adduct for the preparation of a reagent and/or kit for diagnosing, monitoring or detecting exposure of pyrrolizidine alkaloids of a subject of interest, said diagnosis, monitoring or detection comprising:

(i) Measuring the concentration or amount of pyrrole-amino acid adducts in a biological sample obtained from the target subject;

(ii) Comparing the measured concentration or amount of the pyrrole-amino acid adduct to the concentration or amount of a reference control; and

(iii) When the measured concentration or amount of the pyrrole-amino acid adduct in the sample is higher than the concentration or amount of the reference control, then it is considered that pyrrolizidine alkaloid exposure in the target subject is detected;

wherein the pyrrole-amino acid adduct is selected from One or more of them, or selected fromOne or more of (a) and +.>Is a combination of (a);

the biological sample is selected from the group consisting of blood and urine; and

the reference control is the concentration or amount of the pyrrole-amino acid adduct measured in a biological sample obtained in a subject without pyrrolizidine alkaloid exposure.

2. The use of claim 1, wherein step (i) comprises measuring by a combined separation-detection assay selected from LC-MS or LC-MS/MS.

3. The use of any one of claims 1-2, wherein step (i) comprises quantifying the concentration or amount of the pyrrole-amino acid adduct measured in the biological sample using a standard calibration curve.

4. The use of any one of claims 1-2, wherein when pyrrolizidine alkaloid exposure is detected in the target subject, the diagnosing, monitoring or detecting further comprises repeating step (i) at a later time using the same type of biological sample obtained from the target subject.

5. The use of claim 4, wherein an increase in the concentration or amount of the pyrrole-amino acid adduct at a later time as compared to the original step (i) is indicative of worsening of the pyrrolizidine alkaloid exposure and a decrease is indicative of improvement of the pyrrolizidine alkaloid exposure.