Diagnosis of liver disease

Abstract

The present invention relates to methods for the detection, diagnosis or prognosis of a liver disease and related methods, compositions and kits. The method relates to the administration of a unique combination of exogenous VOCs which demonstrate an improved accuracy of disease classification compared to using a single exogenous VOC, as the administered exogenous VOCs each display distinct time resolution.

Description

[0001] Diagnosis of Liver Disease Field of Invention The present invention relates to methods for the detection, diagnosis or prognosis of a liver disease and related methods, compositions and kits. Background Currently, three-quarters of people with liver disease are diagnosed late in a hospital setting. Disease progression is often asymptomatic with up to 75% of patients diagnosed at the decompensated stage. The rising prevalence of both acute and chronic liver disease highlights the need for diagnostics that distinguish between early- and late-stage liver disease. In recent years, there has been a growing recognition of the potential of breath analysis in the field of medical diagnostics1. The human breath carries a wealth of information, including gasses and volatile organic compounds (VOCs), which are generated from various physiological processes within the body1. These molecules are influenced by disease states that alter their abundance in the breath2. This phenomenon has led to the identification of specific compounds that can serve as biomarkers for various diseases3. One intriguing facet of breath analysis lies in its non-invasive nature. Unlike traditional diagnostic methods that often require invasive procedures and complex sample collections, breath analysis offers an attractive alternative1,2,4,5. By harnessing the unique metabolic information contained within exhaled breath, clinicians can potentially detect diseases swiftly and accurately, enhancing the overall diagnostic process. For example, conditions such as Small Intestinal Bacterial Overgrowth (SIBO) and Intestinal Methanogen Overgrowth (IMO) – both implicated in gut discomfort – can be diagnosed through the measurement of exhaled hydrogen and methane6. A number of food flavourings which are widely utilized in the food industry are metabolized in the liver and exhaled in the breath, meaning that their abundance depends on the hepatic metabolic capacity5,7,8. These compounds have gained significant attention as potential biomarkers for non-invasive chronic liver disease where alterations in metabolite profiles can offer valuable insights into disease diagnosis, progression, and severity4,9. One such supplement, limonene, has been found associated with cirrhosis in multiple studies7,9-14. Notably, the severity of cirrhosis and individual dietary exposures exert significant influence on the levels of these compounds in the breath4,9,10. Moreover, while limonene has been extensively studied, recent research has indicated that a combination of multiple compounds, rather than a singular biomarker, enhances the accuracy and reliability of cirrhosis diagnosis9. Despite these advancements, few studies have been conducted to validate these findings across diverse patient cohorts. Additionally, the variability in the breath profiles of these compounds across different populations are often influenced by dietary habits and environmental exposures, which poses a significant challenge in identifying biomarkers suitable for a test in the general population. The invention described herein aims to address this need. The inventors have identified a unique combination of exogenous VOC probes that can be used to assess functioning of the liver across different patient cohorts and to distinguish between early- and late-stage liver disease to inform the most appropriate treatment option. Summary of the Invention In one aspect, the invention relates to a method for detecting, diagnosing, staging, monitoring or prognosing a liver disease in a subject, the method comprising: (a) administering two or more exogenous Volatile Organic Compound (VOC) to the subject and measuring the concentration of the administered exogenous VOC and / or metabolite thereof in a biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and optionally (b) measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject. In a second aspect, the invention relates to a method for determining efficacy of a treatment in a subject diagnosed with a liver disease, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject before initiation of the treatment or therapy, and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a first biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, measuring the concentration of one or more endogenous bioproduct of a liver enzyme in the biological sample obtained from said subject; and (b) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject after initiation of the treatment or therapy, and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a second biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject. In a third aspect, the invention relates to a method for assessing the health of a liver in advance of and / or after a surgical procedure performed in a subject, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, (b) measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject. In one embodiment three, four, or five exogenous VOCs are administered to the subject. In one embodiment, the metabolite is selected from one or more of alpha-pinene, alpha- phellandrene, 2-pentanol, 2-methyl furan and / or 2-butanone. In one embodiment, the exogenous VOCs are administered in combination with nonanal and / or benzyl alcohol. In one embodiment, the metabolite is further selected from 1-nonanol, octane, and / or benzaldehyde. In one embodiment, the endogenous bioproduct of is dimethyl selenide. In one embodiment, the biological sample obtained from a subject is obtained prior to the administration of exogenous VOCs, and at multiple time points post administration. In one embodiment, the biological sample obtained from the subject is obtained between 0 and 24 hours post administration of exogenous VOCs. In one embodiment, the method further comprises comparing the concentration of the compounds measured in the biological sample obtained from the subject to the concentration of the same compounds measured in a biological sample obtained from a healthy subject, wherein a difference in the concentration of measured compounds, the ratio between substrate and bioproduct, the ratio between administered VOCs, the ratio between different metabolites, and / or ratio with a standard compound indicates a likelihood of a liver disease. In one embodiment, the concentration of the measured compounds in the biological sample obtained from the test subject are elevated or reduced compared to the concentration of said compounds in a biological sample obtained from a healthy subject. In one embodiment, the ratio between substrate and bioproduct is measured, wherein the substrate is selected from one of limonene, 2-pentanone and, 2-butanol and the bioproduct is selected from one of alpha-pinene, alpha-phellandrene, 2-pentanol, 2-methyl furan and / or 2- butanone. In one embodiment, the method further comprises comparing the ratio between the concentration of an administered VOC and a physiological characteristic of the subject to the ratio between the concentration of the same administered VOC and a physiological characteristic of a healthy subject, wherein a difference in the ratio between the concentration of an administered VOC and a physiological characteristic indicates a likelihood of a liver disease. In one embodiment, the administered VOC is selected from one or more of limonene, 2- pentnaone and / or 2-butanol, and the physiological characteristic is selected from one or more of the subject’s Body Mass Index, Body Surface Area, height, weight, sex, age and / or blood volume. In one embodiment, the biological sample is selected from breath, urine, blood, serum, and / or tissue. In one embodiment, the biological sample is exhaled breath. In one embodiment, the exogenous VOCs are administered at a dose of between 0.1 to 1000 mg. In one embodiment, the administered exogenous VOCs are administered simultaneously, sequentially or separately. In one embodiment, the administered exogenous VOC is formulated as a liquid, a tablet or capsule for fast release. In one embodiment, the administration of the exogenous VOCs is after a fasting period. In one embodiment, the subject has fasted for 1-12 hours. In one embodiment, the administration of exogenous VOCs is oral or parenteral. In one embodiment, the liver disease is a liver disease associated with cirrhosis, portal hypertension, liver fibrosis, metabolic dysfunction-associated steatotic liver disease (MASLD), alcoholic liver disease (ALD), viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, Wilson’s disease, alpha-1 antitrypsin deficiency, drug-induced liver injury, acetaminophen toxicity, hepatic encephalopathy, Budd-Chiari syndrome, primary biliary cholangitis, primary sclerosing cholangitis, and / or haemochromatosis. In one embodiment, the cirrhosis is compensated or decompensated. In one embodiment, the exogenous VOC, metabolite and / or endogenous bioproduct is labelled. In one embodiment, the label is deuterium. In one embodiment, the concentration of compounds is measured by spectrometry. In one embodiment, the spectrometry is gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry. In one embodiment, the invention relates to a method for determining liver function comprising a method as described herein. In one embodiment, the invention relates to a method for triaging a subject with liver disease comprising a method as described herein. In one embodiment, the invention relates to a method for identifying a subject with cirrhosis suitable for causative treatments comprising a method as described herein. In one embodiment, the causative treatment is selected from viral suppression / eradication, alcohol abstinence, lifestyle improvements, and / or early treatment of precipitating risk factors including dehydration, hypotension, and / or infections. In one embodiment, the invention relates to a method for identifying a subject suitable for advanced management treatment comprising a method as described herein. In one embodiment, the advanced management treatment is selected from fluid resuscitation, vasopressors, endoscopic band ligation, injection sclerotherapy, trans-jugular intrahepatic portosystemic shunt (TIPS), paracentesis, lactulose and / or liver transplant. In one embodiment, the invention relates to a method indicating the likelihood of liver cirrhosis according to a method described herein. In one embodiment, the method further comprises the generation of a risk score based on the combination of quantitative measurements of exogenous VOCs and / or endogenous bioproducts from an exhaled breath sample. In one embodiment, the quantitative measurements of exogenous VOCs and / or endogenous bioproducts from an exhaled breath sample is combined with demographic and / or clinical metrics to generate the risk score. In a fourth aspect, the invention relates to a composition comprising one or more exogenous VOC, wherein the VOC is selected from limonene, 2-pentanone and / or 2-butanol. In one embodiment, the composition additionally contains nonanal and / or benzyl alcohol. In one embodiment, the invention relates to a kit comprising the composition as described herein, and a device for capturing a breath sample from a subject. Figures The invention is further described in the following non-limiting figures. Figure 1. Representation of the effect of liver cirrhosis on compounds pharmacokinetics based on hepatic extraction. Figure 2. Volcano plot generated comparing healthy controls with subjects with cirrhosis in the Chile cohort. In orange are highlighted the VOCs that are significantly different between the two groups. Figure 3. ROC plot for (A) Chile and (B) UK cohort. Figure 4. Amount of deuterated (A) 2-Butanol and (B) 2-Butanone in the tested experimental conditions which consist in controls: vehicle + hepatocytes (Vehicle + Hep), Substrates only; and the sampling condition: Substrates + Hepatocytes (Substrates + Hep). Figure 5. Amount of deuterated (A) 2-Pentanone and (B) 2-Pentanol in the tested experimental conditions which consist in controls: vehicle + hepatocytes (Vehicle + Hep), Substrates only; and the sampling condition: Substrates + Hepatocytes (Substrates + Hep). Figure 6. Amount of deuterated (A) nonanal and (B) 1-nonaol in the tested experimental conditions which consist in controls: vehicle + hepatocytes (Vehicle + Hep), Substrates only; and the sampling condition: Substrates + Hepatocytes (Substrates + Hep). Figure 7. Amount of deuterated (A) benzyl alcohol and (B) benzaldehyde in the tested experimental conditions which consist in controls: vehicle + hepatocytes (Vehicle + Hep), Substrates only; and the sampling condition: Substrates + Hepatocytes (Substrates + Hep). Figure 8. Amount of deuterated limonene in the tested experimental conditions which consist in controls: vehicle + hepatocytes (Vehicle + Hep), Substrates only; and the sampling condition: Substrates + Hepatocytes (Substrates + Hep). Figure 9. Breath profile of investigated compounds. The administered compound 2-butanol is shown in (A)(i), and the bioproduct 2-butanone is shown in (A)(ii). The administered compound 2-pentanone is shown in (B)(i), and the bioproduct 2-pentanol is shown in (B)(ii). The administered compound limonene is shown in (C)(i), and the bioproducts alpha-pinene and alpha-phellandrene are shown in (C)(ii) and (C)(iii), respectively. The administered compound benzyl alcohol is shown in (D)(i), and the benzaldehyde is shown in (D)(ii). The administered compound nonanal is shown in (E)(i), and the bioproducts 1-nonanol and octane are shown in (E)(ii) and (E)(iii), respectively. Figure 10. Roc plots showing area under the ROC curve before exogenous volatile organic compounds (EVOCs) administration at 0 minutes, and 20 minutes after administration. All the timepoints post administration showed an AUC > 0.8. Figure 11. EVOCs and metabolic pathways targeted by LIBRA.2-pentanone and 2-butanol are metabolized to respectively 2-pentanol and 2-butanone by alcohol dehydrogenase, while limonene is metabolized by CYP2C9 and CYP2C19 to more soluble compounds excreted in the urines. Figure 12. ROC plot of classification model at baseline. Figure 13. ROC plot of classification model at 15 minutes. Figure 14. ROC plot of classification model at 30 minutes. Figure 15. Boxplot showing detection of limonene at 0 min (A), 15, min (B), and 30 min (C). Figure 16. Boxplot showing detection of 2-pentanone at 0 min (A), 15, min (B), and 30 min (C), and 2-pentanol at 0 min (D), 15, min (E), and 30 min (F). Figure 17. Boxplot showing detection of 2-butanol at 0 min (A), 15, min (B), and 30 min (C), and 2-butanone at 0 min (D), 15, min (E), and 30 min (F). Detailed Description The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). The present invention relates to methods for the detection, diagnosis or prognosis of a liver disease and related methods, compositions and kits. According to one aspect of the invention, there is provided a method for detecting, diagnosing, staging, monitoring or prognosing a liver disease in a subject, the method comprising: (a) administering two or more exogenous Volatile Organic Compound (VOC) to the subject and measuring the concentration of the administered exogenous VOC and / or metabolite thereof in a biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and optionally (b) measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject. The inventors have surprisingly found that the unique combination of exogenous VOCs of the present invention demonstrate an improved accuracy of classification compared to using a single exogenous VOC. The present invention describes the measurement of at least two administered exogenous VOCs and / or metabolites thereof in a biological sample obtained from a subject at multiple timepoints post administration. The inventors have shown that the unique combination of exogenous VOCs of the present invention displays different classification performance at multiple timepoints post administration, i.e. the administered exogenous VOCs each display distinct time resolution. This is advantageous as the method of the present invention has increased robustness and improved the threshold for accurate disease classification. This also reduced the potential for false positive and / or false negative results. The term VOC refers to any compound of carbon, excluding carbon monoxide, carbon dioxide, carbonic acid, metallic carbides or carbonates and ammonium carbonate, which participates in atmospheric photochemical reactions. Thus, the VOC is not carbon dioxide or carbon monoxide. Generally, VOCs are defined as organic chemical compounds whose composition makes it possible for them to evaporate under normal indoor atmospheric conditions of temperature and pressure. Since the volatility of a compound is generally higher the lower its boiling point temperature, the volatility of organic compounds is sometimes defined and classified by their boiling points. In one embodiment, a VOC is any organic compound having an initial boiling point less than or equal to about 250° C measured at a standard atmospheric pressure of about 101.3 kPa. The VOC that is measured according to the methods is not endogenous to the subject. This ensures that any readings are not contaminated by endogenous VOC that are naturally produced. The term “exogenous VOC” (EVOC) is a VOC that can be administered to a subject that is metabolised by an enzyme within the subject. An exogenous VOC refers to a chemical compound that is recognized by the enzyme of interest and for which the enzyme catalyses conversion of the VOC into a different chemical compound which is referred to herein as a "metabolite". An “exogenous VOC” is a xenobiotic, i.e., a substance that is foreign to the subject’s body and which is specifically and selectively metabolised by the enzyme in vivo. The term “endogenous bioproduct” refers to a naturally occurring metabolite produced by the subject and excreted into a biological matrix. The subject may be a subject that has been diagnosed with a liver disease. The subject may be a subject that is predicted to have a liver disease. The term “prognosis” refers to the forecast or likely outcome of a disease. As used herein, it refers to the probable outcome of liver disease, including whether the disease will respond to treatment or mitigation efforts and / or the likelihood that the disease will progress. The method described herein may diagnose or detect a liver disease in a subject that may be at risk of reduced liver function. By “at risk of reduced liver function” is meant that a subject has, or has been exposed to, one or more risk factors for liver disease, i.e., one or more factors that are known to increase the likelihood of a subject developing a liver disease. For example, the patient may have one or more risk factors selected from, but not limited to, obesity, alcoholism, family history of liver disease, type 2 diabetes, dyslipidaemia, bowel disease, a genetic predisposition to a liver disease and / or any other disease, syndrome and / or pathology that result in an increased likelihood of gut-related problems. In such embodiments, the patient typically has not previously been diagnosed with a liver disease. In one embodiment, the liver disease is not non-alcoholic steatohepatitis (NASH). In a second aspect, the invention relates to a method for determining efficacy of a treatment in a subject diagnosed with a liver disease, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject before initiation of the treatment or therapy, and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a first biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, measuring the concentration of one or more endogenous bioproduct of a liver enzyme in the biological sample obtained from said subject; and (b) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject after initiation of the treatment or therapy, and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a second biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject. The second biological sample may be taken at any suitable time point after initiation of the treatment or therapy. For example, the second biological sample may be taken hours, days or weeks after the initiation of the treatment or therapy. A further biological sample, such as a third, fourth, fifth, etc. biological sample, may be taken at a different time point to the second biological sample. It will be understood by the skilled person that taking two or more biological samples after initiation of the treatment or therapy may enable monitoring of the efficacy of a treatment or therapy over time. Alternatively or additionally, the efficacy of a treatment or therapy may be monitored in real-time. For example, the efficacy of a treatment or therapy may be measured in real-time by selected ion flow tube mass spectrometry (SiFT-MS). Depending on the compound, if the concentration increases or decreases compared to the concentration of the compound obtained in the first sample, the therapy is considered to be effective. An effective treatment or therapy may be continued, or discontinued if the patient’s condition has improved and is no longer in need of treatment. An ineffective treatment may be altered or modified, or replaced with other treatment. The treatment may comprise surgery or at least one drug selected from statins, incretin analogues, metformin, rimonabant, thiazolidinediones, and orlistat. In order to determine the efficacy of treatment multiple samples may be obtained at various times points. As such the method may comprise analysing a first biological sample obtained from said subject at a first time point, and then analysing one or more additional biological samples obtained from said subject at one or more additional time points or ratios thereof. Thus, the method may also comprise the step of administering a treatment. When the liver disease is NASH, said treatment may be gastric bypass surgery, and / or a drug-based treatment comprising the administration of at least one drug selected from statins, incretin analogues, metformin, rimonabant, thiazolidinediones, and orlistat. In a third aspect, the invention relates to a method for assessing the health of a liver in advance of and / or after a surgical procedure performed in a subject, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, (b) measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject. In one embodiment, two, three, four, or five exogenous VOCs are administered to the subject. The product of the metabolism of the administered exogenous VOCs can be used to assess the metabolic activity of liver enzymes to make inferences about the physiological state of the body or health of an individual. In one embodiment, the liver enzyme may be an alcohol dehydrogenase, such as αα-alcohol dehydrogenase. In one embodiment, the liver enzyme may be an aldo-keto-reductase, such as aldo-keto-reductase-B10. In one embodiment, the liver enzyme may be a cytochrome P450 (CYP). For example, the exogenous VOC 2-butanol is a substrate for the liver enzyme αα-alcohol dehydrogenase, which produces the metabolite 2- butanone. 2-butanone is a substrate for CYP, which produces the metabolites 3-hydroxy-2- butanone and 2,3-butanediol. The metabolism of the exogenous VOC limonene produces the metabolites alpha-pinene and / or alpha-phellandrene. The exogenous VOC 2-pentanone is a substrate for the liver enzyme αα-alcohol dehydrogenase, which produces the metabolites 2- pentanol and / or 2-methyl furan. Thus, in one embodiment, the metabolite that is measured in the method of the present invention is further selected from alpha-pinene, alpha-phellandrene, 2-pentanol, 2-methyl furan and / or 2-butanone. The exogenous VOC limonene can also be metabolised by the liver enzyme CYP2C9 or CYP2C19 to produce perillyl alcohol and trans-carveol, respectively. In one embodiment, the exogenous VOCs are administered in combination with nonanal and / or benzyl alcohol. In one embodiment, limonene and 2-pentanone are administered to the subject. In one embodiment, limonene and 2-butanol are administered to the subject. In one embodiment, limonene and nonanal are administered to the subject. In one embodiment, limonene and benzyl alcohol are administered to the subject. In one embodiment, 2-pentanone and 2-butanol are administered to the subject. In one embodiment, 2-pentanone and nonanal are administered to the subject. In one embodiment, 2-pentanone and benzyl alcohol are administered to the subject. In one embodiment, 2-butanol and nonanal are administered to the subject. In one embodiment, 2-butanol and benzyl alcohol are administered to the subject. In one embodiment, limonene, 2-pentanone and 2-butanol are administered to the subject. In one embodiment, limonene, 2-pentanone and nonanal are administered to the subject. In one embodiment, limonene, 2-pentanone and benzyl alcohol are administered to the subject. In one embodiment, limonene, 2-butanol and nonanal are administered to the subject. In one embodiment, limonene, 2-butanol and benzyl alcohol are administered to the subject. In one embodiment, limonene, nonanal and benzyl alcohol are administered to the subject. In one embodiment, 2-pentanone, 2-butanol and nonanal are administered to the subject. In one embodiment, 2-pentanone, 2-butanol and benzyl alcohol are administered to the subject. In one embodiment, 2-pentanone, nonanal and benzyl alcohol are administered to the subject. In one embodiment, 2-butanol, nonanal and benzyl alcohol are administered to the subject. In one embodiment, limonene, 2-pentanone, 2-butanol and nonanal are administered to the subject. In one embodiment, limonene, 2-pentanone, 2-butanol and benzyl alcohol are administered to the subject. In one embodiment, limonene, 2-pentanone, nonanal and benzyl alcohol are administered to the subject. In one embodiment, limonene, 2-butanol, limonene, and 2-pentanone are administered to the subject. In one embodiment, 2-pentanone, 2-butanol, nonanal, and benzyl alcohol are administered to the subject. In one embodiment, limonene, 2-pentanone, 2-butanol, nonanal and benzyl alcohol are administered to the subject. The exogenous VOC nonanal is a substrate for the liver enzyme aldo-keto-reductase-B10, which produces the metabolite 1-nonanol and / or octane. The exogenous VOC benzyl alcohol is a substrate for the liver enzyme alcohol dehydrogenase, which produces the metabolite benzaldehyde. Thus, in one embodiment, the metabolite that is measured in the method of the present invention is selected from one or more of 1-nonanol, octane and / or benzaldehyde. In one embodiment, the endogenous bioproduct of is dimethyl selenide. Dimethyl selenide is one of the excretion products of selenium metabolism. Impaired organic and inorganic selenium biotransformation in cirrhotic patients results in downregulation of dimethyl selenide. In one embodiment, the method of the present invention may comprise the administration of dimethyl selenide as an exogenous VOC. The specific combination of administered exogenous VOCs of the present invention provides a more accurate and robust method for detecting liver disease in a subject. The inventors have surprisingly found that the timepoints at which the concentration of administered exogenous VOCs and their substrates are measured is crucial for the classification of biological samples. Thus, in one embodiment, the biological sample obtained from a subject is obtained prior to the administration of exogenous VOCs, and at multiple time points post administration. The biological sample may be obtained at any time point prior to administration of exogenous VOCs, such as about 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 30 seconds before the administration of exogenous VOCs. The time point may also be earlier or later. The biological sample may be obtained at any time point post administration of exogenous VOCs, such as about 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 15 hours, 18 hours. 21 hours, 24 hours, 36 hours, 48 hours after the administration of exogenous VOCs. The time point may also be earlier or later. In one embodiment, the method further comprises comparing the concentration of the compounds measured in the biological sample obtained from the subject to the concentration of the same compounds measured in a biological sample obtained from a healthy subject, wherein a difference in the concentration of measured compounds, the ratio between substrate and bioproduct, the ratio between administered VOCs, the ratio between different metabolites and / or ratio with a standard compound indicates a likelihood of a liver disease. The ratio between administered VOCs may be measured. The administered VOCs may be any exogenous VOC disclosed herein. The ratio between substrate and bioproduct may be measured. The substrate may be selected from any substrate disclosed herein and the bioproduct may be selected from any one of bioproduct disclosed herein. In one embodiment, the method further comprises comparing the ratio between the concentration of an administered VOC and a physiological characteristic of the subject to the ratio between the concentration of the same administered VOC and a physiological characteristic of a healthy subject, wherein a difference in the ratio between the concentration of an administered VOC and a physiological characteristic indicates a likelihood of a liver disease. The administered VOC may be any exogenous VOC disclosed herein, and the physiological characteristic may be selected from one or more of the subject’s Body Mass Index, Body Surface Area, height, weight, sex, age and / or blood volume. In one embodiment, the method further comprises the generation of a model including multiple adjusting weighting factors. The model may account for differences in disease aetiology and / or demographic factors Body Mass Index, Body Surface Area, height, weight, sex, age and / or blood volume. Errors in dosage and / or administration of exogenous VOCs need to be accounted for to ensure correct identification of a subject with liver disease. Therefore, in one embodiment, the value of a measured concentration of one or more administered exogenous VOCs and / or metabolite thereof may be used to normalise the value of other administered exogenous VOCs and / or metabolites thereof. This lowers the threshold for disease classification to ensure the method accounts for differences in disease aetiology, body mass, age, and / or errors in exogenous VOC administration. When the biological sample is exhaled breath, the concentration of the compounds measured in the biological sample may be at least 0.0001 mmol / L, such as 0.0002, 0.0003, 0.0004, 0.0005. 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009 or 0.01 mmol / L. In one embodiment, the concentration of the measured compounds in the biological sample obtained from the test subject are elevated or reduced compared to the concentration of said compounds in a biological sample obtained from a healthy subject. A difference in the concentration of the measured compounds between the test subject and the healthy subject indicates a likelihood of liver disease. A “likelihood of a liver disease” means that the probability that the liver disease exists in the subject specimen is about 50% or more, for example 60%, 70%, 80% or 90%. When the concentration of compounds is measured from a healthy subject, an increased concentration of one, more or each of 2-butanol, nonanal, benzyl alcohol, 2-butanone, 1- nonanol, octane and / or benzaldehyde, limonene, 2-pentanone, alpha-pinene alpha- phellandrene, 2-pentanol, 2-methyl furan e.g., 5%, 10%, 20%, 30%, 40%, 50% or more, in the test subject values compared to the healthy subject values may indicate a diagnosis of a liver disease, such as cirrhosis, or a risk that the subject will develop a liver disease, such as cirrhosis, or a complication of liver disease, such as decompensated cirrhosis. When the concentration of compounds is measured from a healthy subject, a decreased concentration of dimethyl selenide, e.g. 5%, 10%, 20%, 30%, 40%, 50% or more, in the test subject values compared to the healthy subject values may indicate a diagnosis of a liver disease, such as cirrhosis, or a risk that the subject will develop a liver disease, such as cirrhosis or a complication of liver disease, such as decompensated cirrhosis. When an appropriate reference is indicative of a subject being free of a liver disease a detectable difference (e.g., a statistically significant difference) between the set of healthy subject values and the appropriate reference may be indicative of cirrhosis in the test subject. When an appropriate reference is indicative of a liver disease, a lack of a detectable difference (e.g., lack of a statistically significant difference) between the healthy subject values and the appropriate reference may be indicative of cirrhosis in the test subject. Thus, the methods may include diagnosing the subject as having a likelihood or increased risk of a liver disease if the level of one or more of compounds 2-butanol, nonanal, benzyl alcohol, 2-butanone, 1-nonanol, octane and / or benzaldehyde, limonene, 2-pentanone, alpha-pinene alpha-phellandrene, 2-pentanol, 2-methyl furan or dimethyl selenide is different from the level of a healthy subject value. Additionally or alternatively, the methods may include diagnosing the subject as having a likelihood or increased risk of a liver disease if the level of one or more of compounds i) to vii) is the same, substantially the same or different from the level of a reference subject value, wherein the reference subject value is obtained from a subject that has been diagnosed with a liver disease. The term "deviation of the amount" refers either to elevated or reduced amounts of one or more of compounds i) to vii) in a biological sample from a subject compared to the reference subject values. By "elevated amounts" we mean that the amount of said one or more of compounds i) to vii) in a biological sample from a subject is statistically higher than the corresponding reference subject value. By "reduced amounts" we mean that the amount of one or more of compounds i) to vii) in a biological sample from a subject is statistically lower than the corresponding reference subject value. The amount may be considered to be statistically higher or lower if its value differs from a predetermined threshold value. This threshold value can, for example, be the median of the amount of the compound determined in a biological sample from a population of healthy subjects. The term "no deviation of the amount" refers to similar or unchanged amounts of one or more of compounds i) to vii) in a sample of exhaled breath from a subject compared to the corresponding reference subject value. By "similar or unchanged level" is meant that the difference of the amount of said one or more of compounds i) to vii) in a biological sample from the subject compared to the corresponding reference subject value is not statistically significant. Preferably, the reference subject value is obtained in samples of exhaled breath obtained from one or more subjects of the same species and the same sex and age group as the subject in which a liver disease is to be determined, prognosed or monitored. Alternatively, the reference subject value may be a previous value for the amount of one or more of compounds i) to vii) obtained in a sample of exhaled breath from a specific subject. This kind of reference value may be used if the method is to be used for monitoring the liver disease, e.g., over time, or to monitor the response of a subject to a particular treatment. The term “biological sample” may be used interchangeably with the term biological matrix. The biological sample or matrix may be selected from breath, stool, urine, blood, serum, and / or tissue. The biological sample may be a tissue sample such as adipose tissue, liver, brain, bone marrow, muscle or hair. The biological sample may be a sample of bodily fluid. Methods are well known in the art for obtaining bodily fluid samples. The bodily fluid sample may be a sample of blood, stool, urine or exhaled breath. The sample of blood may comprise one or more of blood plasma, red blood cells, white blood cells, platelets. The blood sample may comprise any combination of blood plasma, red blood cells, white blood cells, platelets. The biological sample may be hair, blood, stool, urine and / or exhaled breath, for example blood, urine and / or exhaled breath, for example blood, urine and / or exhaled breath. The biological sample may be exhaled breath. Where the biological sample is a sample of exhaled breath, the breath sample can include air exhaled from one or more different parts of the patient’s body (e.g. nostrils, pharynx, trachea, bronchioles, alveoli etc.). For the collection of a breath sample and methods of measurement, the device and methods described in WO2017 / 187120 or WO2017 / 187141 (both publications are hereby incorporated by reference) can be used. In embodiments wherein the biological sample is a sample of exhaled breath, this may be obtained by collecting exhaled air from the patient, for example by requesting the subject to exhale air into a gas-sampling container, such as a bag, a bottle or any other suitable gas- sampling product. Preferably the gas-sampling container resists gas permeation both into and out of the bag and / or is chemically inert, thereby assuring sample integrity. Exhaled breath may also be collected using a breath collector apparatus. Preferably, collection of a sample of exhaled breath is performed in a minimally invasive or a non-invasive manner. In one embodiment, a biological sample is obtained from a subject and the sample is analysed in vitro or ex vivo. In one embodiment, the exogenous VOCs are administered at a dose of between 0.1 to 1000 mg. Typically, the amount of an exogenous VOC administered as part of the methods of the invention is at least about 0.01% of the substrate by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject's body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the subject's body weight, and more preferably from about 1 mg / kg to about 10 mg / kg of the subject's body weight. In one embodiment, the administered exogenous VOCs are administered simultaneously, sequentially or separately. The administration of the exogenous VOCs of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or subcutaneously. When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the exogenous VOCs for administration can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose, sucralose, or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the exogenous VOCs are in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil. The exogenous VOCs for administration can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. Compositions can take the form of one or more dosage units. It may be desirable to administer the composition by intravenous injection or infusion. In one embodiment, the administration of the exogenous VOCs is after a fasting period. In another embodiment, the subject may be fasting overnight and fasting can be combined with the provision of the substrate as a liquid or fast release / dissolving tablet or fast release / dissolving capsule or other oral administration format. In one embodiment, the subject has fasted for 1-12 hours. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered orally or parenterally. In one embodiment, the exogenous VOCs are administered intravenously, subcutaneously, intramuscularly, or intradermally. The liver disease may be any suitable liver disease. The liver disease may be portal hypertension, viral hepatitis, metabolic-associated steatotic liver disease (MASLD), alcoholic liver disease, alcoholic hepatitis, autoimmune hepatitis, Wilson’s disease, alpha-1 antitrypsin deficiency, drug-induced liver injury, acetaminophen toxicity, hepatic encephalopathy, Budd- Chiari syndrome, primary biliary cholangitis, primary sclerosing cholangitis, haemochromatosis, or a combination of these diseases . In one embodiment, the liver disease is a liver disease associated with cirrhosis. The term “cirrhosis,” “liver cirrhosis” or “hepatic cirrhosis” refers to a condition in which the liver does not function properly due to long-term damage. This damage is characterized by the replacement of normal liver tissue by scar tissue (i.e. fibrosis). The disease generally develops slowly over months or years, often with no symptoms. Eventually, excessive scar formation will result in loss of liver function. Patients with cirrhosis may be asymptomatic or symptomatic, depending on whether the cirrhosis is compensated or decompensated, respectively. Thus, in one embodiment, the cirrhosis is compensated or decompensated. In one embodiment, the method described herein relates to a method of prognosing cirrhosis progression. The distinction between compensated and decompensated cirrhosis dictates the optimum treatment plan for a subject with cirrhosis. In one embodiment, the invention relates to a method for triaging a subject with liver disease comprising a method as described herein. Damage to the liver is often permanent, and interventions during the early stages of liver disease may limit complications. Compensated liver cirrhosis may be reversible if the underlying cause is treated before the disease progresses. Thus, in one embodiment, the invention relates to a method for identifying a subject with cirrhosis suitable for causative treatments comprising a method as described herein. The causative treatment may be selected from viral suppression / eradication, alcohol abstinence, lifestyle improvements, and / or early treatment of precipitating risk factors including dehydration, hypotension, and / or infections. The transition from compensated to decompensated cirrhosis may be of characterised by the onset of one or more of ascites, jaundice, hepatic encephalopathy, variceal bleeding, and / or hepatocellular carcinoma. In one embodiment, the invention relates to a method for identifying a subject suitable for advanced management treatment comprising a method as described herein. The advanced management treatment may be selected from fluid resuscitation, vasopressors, endoscopic band ligation, injection sclerotherapy, trans-jugular intrahepatic portosystemic shunt (TIPS), paracentesis, lactulose and / or liver transplant. In one embodiment, the liver disease is portal hypertension and / or liver fibrosis. In one embodiment, the exogenous VOC, metabolite and / or endogenous bioproduct is labelled. The label may be deuterium. The label may be an isotope label, for example 12C, 13C, 14C, 2H, 14N or 18O. In one embodiment, the exogenous VOC is not labelled. The determination of the amount of an administered exogenous VOC, a metabolite thereof and / or an endogenous bioproduct in a sample of exhaled breath from a subject may be performed by the use of at least one technique including, but not limited to, Gas-Chromatography (GC), Gas-Chromatography-lined Mass Spectrometry (GC / MS), Liquid Chromatography- tandem mass spectrometry (LC / MS), Ion Mobility Spectrometry / Mass Spectrometry (IMS / MS), Proton Transfer Reaction Mass-Spectrometry (PTR-MS), Electronic Nose device, quartz crystal microbalance or chemically sensitive sensors. The amount of an administered exogenous VOC, a metabolite thereof and / or an endogenous bioproduct in a sample of exhaled breath from a patient may be determined using thermal desorption-gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). In certain embodiments, breath of the patient is collected in an inert bag, then the content of the bag is transported under standardised conditions onto desorption tubes and VOCs are analyzed by thermally desorbing the content of the tube and then separated by capillary gas chromatography. Then volatile organic peaks corresponding to the compound of interest (i.e., ethanol, one or more sugar fermentation product, such as 2,3-butanediol, and / or a direct ethanol biomarker) are detected with MS and identified using for example a library, such as the National Institute of Standards and Technology. Thermal desorption may be performed at the GC inlet at a temperature of, e.g., about 200-350°C. In all chromatography, separation occurs when the sample mixture is introduced (injected) into a mobile phase. Gas chromatography (GC) typically uses an inert gas such as helium as the mobile phase. GC / MS allows for the separation, identification and / or quantification of individual components from a biological sample. MS methods which may be used with the present invention include, but are not limited to, electron ionization, electrospray ionization, glow discharge, field desorption (FD), fast atom bombardment (FAB), thermospray, desorption / ionization on silicon (DIOS), Direct Analysis in Real Time (DART), atmospheric pressure chemical ionization (APCI), secondary ion mass spectrometry (SIMS), spark ionization and thermal ionization (TIMS). Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is an example of a mass spectroscopy method which may be used to determine the amount of ethanol, one or more sugar fermentation product, such as 2,3-butanediol, and / or a direct ethanol biomarker from a sample of exhaled breath from a patient. The method may comprise collecting different selected exhaled breath samples, or fractions thereof, on a single breath sample capture device. For example, the method may comprise the steps of: (a) collecting a first exhaled breath sample by contacting the sample with a capture device comprising an adsorbent material; (b) collecting a second exhaled breath sample by contacting the second sample with said capture device, wherein the first and second exhaled breath samples are caused to be captured on the capture device in a spatially separated manner. The capture device may comprise an adsorbent material in the form of a porous polymeric resin. Suitable adsorbent materials include Tenax® resins and Carbograph® materials. Tenax® is a porous polymeric resin based on a 2,6-diphenyl-p-propylene oxide monomer. Carbograph® materials are graphitized carbon blacks. In one embodiment, the material is Tenax GR, which comprises a mixture of Tenax® TA and 30% graphite. One Carbograph® adsorbent is Carbograph 5TD. The capture device may comprise both Tenax GR and Carbograph 5TD. The capture device is conveniently a sorbent tube. These are hollow metal cylinders, typically of standard dimensions (3½ inches in length with a ¼ inch internal diameter) packed with a suitable adsorbent material. Where the biological sample is exhaled breath, the method may comprise the steps of A) the subject inhaling purified air and B) the subject providing an exhaled breath sample, and wherein step A) is carried out before step B). Advantageously, the subject inhaling purified air prior to a sample of exhaled breath being taken (for analysis) may minimise environmental contribution to the signal. As such, the methods may comprise the steps of: (a) administering purified air to the subject by inhalation; (b) collecting a sample of exhaled breath from the subject; and (c) measuring the concentration of each of compounds i) to vii) in the sample of exhaled breath obtained from the subject, wherein step (a) is carried out before step (b). In one embodiment, the invention relates to a method for determining liver function comprising a method as described herein. In one embodiment, the invention relates to a method indicating the likelihood of liver cirrhosis according to the method described herein. In one embodiment, the method further comprises the generation of a risk score based on the combination of quantitative measurements of exogenous VOCs and / or endogenous bioproducts from an exhaled breath sample. In one embodiment, the quantitative measurements of exogenous VOCs and / or endogenous bioproducts from an exhaled breath sample is combined with demographic and / or clinical metrics to generate the risk score. The algorithm used to calculate a risk assessment score in a method disclosed herein may group the concentration values of one or more of 2-butanol, nonanal, benzyl alcohol, 2-butanone, 1- nonanol, octane, benzaldehyde, limonene, 2-pentanone, alpha-pinene alpha-phellandrene, 2- pentanol, 2-methyl furan and / or dimethyl selenide, and the risk score can be derived from any algorithm known in the art. The algorithms are sets of rules for describing the risk assessment of a liver disease. The rule set may be defined exclusively algebraically but may also include alternative or multiple decision points requiring domain-specific knowledge, expert interpretation or other clinical indicators. Many algorithms that can provide different risk assessments can be developed using concentration profiles of a suitable substrate and / or metabolite. For example, the risk scores of an individual may be generated using a Cox proportional hazard model. An individual's prognostic categorization can also be determined by using a statistical model or a machine learning algorithm, which computes the probability of recurrence based on the individual's concentration of one or each of compounds 2-butanol, nonanal, benzyl alcohol, 2- butanone, 1-nonanol, octane, benzaldehyde, limonene, 2-pentanone, alpha-pinene alpha- phellandrene, 2-pentanol, 2-methyl furan and / or dimethyl selenide. Based on the determination of a risk, individuals can be partitioned into risk groups (e.g., tertiles or quartiles) based on a selected value of the risk score, where all individuals with values in a given range can be classified as belonging to a particular risk group. Thus, the values chosen will define risk groups of patients with respectively greater or lesser risk. Risk groups can further be classified on different ranges of mortality, for example, on 6 month, 1-year, 2-year, 3-year, 4- year, 5-year, 10-year, 25-year mortality. Risk groups can further be classified on different ranges of events associated with a liver disease. For example, when the liver disease is cirrhosis, the risk groups can be classified on different ranges of events associated with cirrhosis, which can include, but is not limited, likelihood of progression to cirrhosis or cirrhosis with decompensation. The technology described herein may be associated with a programmable machine designed to perform a sequence of arithmetic or logical operations as provided by the methods described herein. For example, some embodiments of the technology are associated with (e.g., implemented in) computer software and / or computer hardware. In one aspect, the technology relates to a computer comprising a form of memory, an element for performing arithmetic and logical operations, and a processing element (e.g., a microprocessor) for executing a series of instructions (e.g., a method as provided herein) to read, manipulate, and store data. Therefore, certain embodiments employ processes involving data stored in or transferred through one or more computer systems or other processing systems. Embodiments also relate to apparatus for performing these operations. This apparatus can be specially constructed for the required purposes, or it can be a general-purpose computer (or a group of computers) selectively activated or reconfigured by a computer program and / or data structure stored in the computer. In some embodiments, a group of processors performs some or all of the recited analytical operations collaboratively (e.g., via a network or cloud computing) and / or in parallel. In some embodiments, a microprocessor is part of a system for determining the concentration of one or more of 2-butanol, nonanal, benzyl alcohol, 2-butanone, 1-nonanol, octane, benzaldehyde, limonene, 2-pentanone, alpha-pinene alpha-phellandrene, 2-pentanol, 2-methyl furan and / or dimethyl selenide; generating standard curves; determining a specificity and / or sensitivity of an assay or marker; calculating an ROC curve; sequence analysis; all as described herein or is known in the art. The amount of each of compounds i) to vii) can be determined by abundance, measured per mole or millimole. The concentration of each of 2-butanol, nonanal, benzyl alcohol, 2-butanone, 1-nonanol, octane, benzaldehyde, limonene, 2-pentanone, alpha-pinene alpha-phellandrene, 2-pentanol, 2-methyl furan and / or dimethyl selenide can be determined by assays known to the skilled person and described herein, including measurements using an optical signal or other measurement known to one of skill. In some embodiments, a microprocessor or computer uses an algorithm to measure the concentration of each of compounds i) to vii). The algorithm can include a mathematical interaction between a marker measurement or a mathematical transform of a marker measurement. The mathematical interaction and / or mathematical transform can be presented in a linear, nonlinear, discontinuous or discrete manner. In some embodiments, a software or hardware component receives the results of multiple assays and determines a single value result to report to a user that indicates a liver disease risk based on the results of the multiple assays. Related embodiments calculate a risk factor based on a mathematical combination (e.g., a weighted combination, a linear combination) of the results from multiple assays as described elsewhere herein. In a second aspect, the invention relates to a composition comprising one or more exogenous VOC, wherein the VOC is selected from limonene, 2-pentanone and / or 2-butanol. The composition may optionally comprise one or more additional active agents, a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. In one embodiment, the composition additionally contains nonanal and / or benzyl alcohol. In one embodiment, there is provided a kit comprising the composition according to one aspect of the invention, a device for capturing a breath sample from a subject. The kit is not limited to these components and further components of it may be apparent to the skilled person. The kit as described herein may include a composition for administration that comprises the exogenous VOCs. This may be formulated as an oral administration, e.g., as a tablet or capsule. The kit may comprise instructions for evaluating or monitoring liver disease in a patient based on the levels of the VOCs and / or metabolites of interest. In some embodiments, the kit contains reagents for measuring the levels of the VOCs and / or metabolites of interest. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed (e.g., sterile, pharmaceutically acceptable buffer and / or other diluents). However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the nucleic acids, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. This component of the kit may be for administration as described above. It may also include a pharmaceutically acceptable carrier or vehicle. The composition can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously. As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Compositions can take the form of one or more dosage units. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety. As used herein, unless otherwise expressly specified, all numbers such as those expressing values, ranges, amounts or percentages may be read as if prefaced by the word "about", even if the term does not expressly appear. Also, the recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1, 2, 3, 4 when referring to, for example, a number of elements, and can also include 1.5, 2, 2.75 and 3.80, when referring to, for example, measurements). The recitation of end points also includes the end point values themselves (e.g. from 1.0 to 5.0 includes both 1.0 and 5.0). Any numerical range recited herein is intended to include all sub-ranges subsumed therein. The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline. The terms "comprising", "comprises" and "comprised of” as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. As used herein, the term "and / or is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Examples The invention is now further described in the non-limiting examples. Example 1. Methods Breath analysis For the Chile cohort, a total of 29 subjects with cirrhosis, and 29 controls were enrolled with a random recruitment. Participants older than 18 years and weighing more than 60 kg were randomly enrolled between January 2022 and December 2022. Cases of cirrhosis were patients under outpatient control with a follow-up in gastroenterology at two centres in Santiago de Chile. Healthy relatives of the patients and other volunteers were invited to participate as controls. For the UK cohort, subjects > 30 years of age were recruited from the clinical research facility at Addenbrooke’s Hospital (Cambridge) or through the Cambridge BioResource between August 2021 and March 2022. All the patients had an established histological or radiological diagnosis of cirrhosis according to EASL and AASLD guidelines15,16. Participants were asked to fast overnight and provide a baseline breath sample before ingestion of an emulsion made of limonene (100 mg), 2-butanol (100 mg), 2-pentanone (100 mg), nonanal (500 mg), benzyl alcohol (500 mg), sucralose (3 mg), Tween 80 (750 mg). Additional breath samples were collected 20-40-60-90-120 minutes post ingestion. The emulsion was prepared by combining the ingredients and mixing for 5 minutes with a blender and used within 24 hours. Acquisition of Breath Biopsy samples was performed in a single room at Clinica Alemana (Santiago, Chile) for the Chile cohort, and Addenbrooke’s Hospital (Cambridge, UK) for the UK cohort. Breath samples were collected by adsorption onto Breath Biopsy Cartridges (made of four ¼” x 3½” inert-coated stainless-steel tubes with Tenax TA / carbograph 5TD adsorbent material (Markes International Ltd, Llantrisant, UK)) through a ReCIVA®Breath Sampler (Owlstone Medical, Cambridge, UK)17. Samples were analyzed using an Orbitrap Exploris 60, a Trace 1310 Gas chromatograph (Thermo Fisher Scientific Inc. Waltham, MA, USA) coupled with TD 100xr thermal desorption system (Markes International Ltd., Llantrisant, UK). Samples were first pre-purged for 1 min at 50 mL / min of Nitrogen, then desorbed at 250 °C for 10 min at the same flow and focused onto a cold trap (U-T12ME-2S, Material Emissions, C4-C32 Markes International Ltd. Llantrisant, UK) at 10 °C. Analytes were then desorbed at 250 °C for 4 min into the GC column (Stabilwax-DA 30 m × 0.25 mm × 0.25 µm, Restek Corporation, Bellefonte, PA, USA) at 2 mL / min helium flow . A split ratio of 2.5:1 was used for baseline (time 0) samples, while a split of 100:1 was used for the timepoints post administration. The GC temperature program was as follows: 40 °C held for 2 min, increasing to 250 °C at a rate of 15 °C / min, and held for 5 min. The ion mass detection was performed using electron ionization (EI) with a mass scan range from m / z 30 to m / z 450 with resolution of 60,000. Electron ionization voltage was set at 70 eV. The ion source and transfer line temperatures were 270 and 250 °C, respectively (instrument settings are in Table 1). Table 1. Instrument settings. General Pre-desorption Standby split flow 50 Pre-purge time (min) 1 (mL / min) Flow path temperature 200 Purge Trap flow (mL / min) 50 (°C) Minimum carrier gas 5 pressure (psi) Tube desorption Desorb Time (min) 10 Desorb temperature 250 (°C) Trap desorb flow 50 (mL / min) Trap settings Trap purge Trap desorption trap low temperature 10 Trap heating rate 1 40 (°C) trap purge time (min) 1 Trap high 1 (°C) 250 trap purge flow 5 Trap Desorb time (min) 4 (mL / min) Trap desorb split On Trap desorb split flow 198 (mL / min) 100:1 GC settings Back inlet setting Gas flow (mL / min) 2 Constant flow Purge flow control 5 (mL / min) GC oven conditions retention time (min) rate target hold time (min) (°C / min) value (°C) 0 Run 2 0 40 2 21 15 250 5 NewRow 21 StopRun Orbitrap settings Ion source temperature 270 (°C) MS transfer line 250 temperature (°C) Electron voltage (eV) 70 Resolution 60,000 C Trap voltage (V) 0 Full MS settings Runtime (min) 0 to 12.5 Polarity Positive Micro scans 1 Resolution 60,000 AGC Target 1.00E+06 Maximum IT auto Scan Range (m / z) 30 – 450 System data type Profile Data about targeted compounds were acquired and exported using Chromeleon (v. 7.2.10, Thermo Fisher Scientific, Waltham, MA, USA), an absolute quantification was obtained by comparison with analytical standards injection: ((R)-(+)-Limonene, 183164, (SigmaAldrich, Gillingham, UK); 2-pentanone, 471194, (SigmaAldrich, Gillingham, UK); dimethyl selenide, 41572, (SigmaAldrich, Gillingham, UK)); 3-Octanone, 46214, (SigmaAldrich, Gillingham, UK); 2- chloro-m-xylene, 276308, (SigmaAldrich, Gillingham, UK); (R)-(-)-2-Pentanol, 44759 (SigmaAldrich, Gillingham, UK); 2 butanol, 294810, (SigmaAldrich, Gillingham, UK); 2-butanone 360473 (SigmaAldrich, Gillingham, UK); Alpha-pinene 147524, (SigmaAldrich, Gillingham, UK); Benzyl Alcohol, W213705, (SigmaAldrich, Gillingham, UK); Benzaldehyde, 378361000, (Thermo Fisher Scientific, MA, USA); Nonanal, 8.0716601, (SigmaAldrich, Gillingham, UK); 1- nonanol, 131210, (SigmaAldrich, Gillingham, UK). For untargeted analysis, data were acquired and exported using Chromeleon (v.7.2.10, Thermo Fisher Scientific, Waltham, MA, USA). Exported data were subsequentially analysed using Compound discoverer (v.3.3, Thermo Fisher Scientific, Waltham, MA, USA) with the following parameters: mass tolerance: 5ppm, TIC threshold: 50000, spectral signal to noise smoothing: 9, ion overlap: 97, RT tolerance: 2 sec., composition threshold: %15, dot product threshold: 500. Compounds were tentatively identified using the National Institute of Standards and Technology (NIST) library and in-house High Resolution Accurate Mass (HRAM) library (https: / / www.owlstonemedical.com / about / blog / 2022 / may / 30 / biomarker-analysis-breath-hram- library / ). Database hits were filtered according to their structure relevance to standard compounds. Identity of compounds was validated by standards injection ((R)-(+)-Limonene, 183164, (SigmaAldrich, Gillingham, UK); dimethyl selenide, 41572, (SigmaAldrich, Gillingham, UK)); 3-Octanone, 46214, (SigmaAldrich, Gillingham, UK); 2-chloro-m-xylene, 276308, (SigmaAldrich, Gillingham, UK). Human suspension hepatocytes A total of 10^6 Human Hepatocytes Suspension, Metabolism Qualified, Male, HMCS1S (Thermo Fisher Scientific, MA, USA) were handled according to supplier instructions, and incubated in 20 ml HiSorb vials (Markes International, UK) in 1 ml complete medium supplemented with 10 ng / µL of the following substrates: 2-butanol-D5 D-1063 (CDN Isotopes, Canada); 2-pentanone- D5 D-0219 (CDN Isotopes, Canada); Limonene-D5 L461754 (CymitQuimica, Spain), Nonanal - D18 D-7078 (CDN Isotopes, Canada); Benzyl alcohol-D7386472 (SigmaAldrich, Gillingham, UK). A total of 5 vials for each experimental condition were assembled. The experimental conditions were hepatocytes with vehicle (0.02% methanol), substrates without hepatocytes, hepatocytes with substrates, hepatocytes with substrates and the alcohol-dehydrogenase (ADH) inhibitor 4-methyl-pyrazole (10 mM) (SigmaAldrich, Gillingham, UK). Vials were incubated for 2 hours in the presence of the HiSorb probes (Markes International, UK) with agitation (300 rpm) at 37° C. At the end of the incubation the HiSorb probes were analysed using gas chromatography-mass spectrometry with the settings described in Table 2.

[0002] Table 2. Gas chromatography-mass spectrometry instrument settings. TD setting 2-3 stage desorb settings General Pre-desorption Standby split flow (mL / min) 50 Pre-purge time (min) 1 Flow path temperature (°C) 200 Purge Trap flow (mL / min) 50 Minimum carrier gas 5 pressure (psi) Tube desorption Desorb Time (min) 10 Desorb temperature (°C) 250 Trap desorb flow (mL / min) 50 Trap settings Trap purge Trap desorption trap low temperature (°C) 10 Trap heating rate 1 40 trap purge time (min) 1 Trap high 1 (°C) 250 trap purge flow (mL / min) 50 Trap Desorb time (min) 4 Trap desorb split on1 Trap desorb split flow 1 20 (mL / min) GC settings gas flow (mL / min) 2 GC oven conditions retention time (min) rate (°C / min) target value (°C) hold time (min) 0 0 40 2 16 15 250 5 21 stop run Orbitrap settings Acquisition time (min) 21 MS transfer line temperature 250 (°C) Ion source temperature (°C) 270 Ionisation mode Electron Ionisation (EI) Electron voltage 70 (eVolt) Polarity Positive AGC Target 1.00E+06 Scan range (m / z) 30-450 Maximum IT auto Resolution 60000 Aux temperature 1 and 2 (°C) 250 The substrates and the following bioproducts or the nearest available deuterated analog were measured: 2-pentanol-D5, D-5139, (CDN Isotopes, Canada); 2-butanone-D5, D-0293, (CDN Isotopes, Canada); 1-nonanol-D18, D-3999, (CDN Isotopes, Canada); Octane-D18, 151971, (SigmaAldrich, Gillingham, UK); alpha-pinene-D3, ALN2123, (EPTES - Analytical Food & Environment, Switzerland); 2-methylfuran-D3, M305441, (LGC standards, UK). Statistical analysis Data were analysed using Python (Python Software Foundation, Fredericksburg, VA, USA, V. 3.11)18and RStudio (Posit, PBC, Boston, MA, USA, V. 2023.06.1)19. Data visualisation was performed using libraries matplotlib (V. 3.7)20, seaborn (V. 0.12.2)21, Corrplot (V. 0.92)22, and ggplot2 (V.3.4.4)23. Intensity of VOCs (count / m), median, and range were compared between experimental conditions using the Mann-Whitney U-test for non-parametric data, and p-values <0.05 were considered statistically significant. Classification models were built as described before. Briefly, within the Chile cohort, the top VOCs for classification were identified by stability selection24and LASSO logistic regression25. Model performance was validated by five-fold cross validation on an 80 / 20% split, with VOC selection performed within each fold training set. Only features above a certain stability score were selected in the model. The importance of each feature for classification was expressed as the resulting average stability score across the five-fold cross validation. Example 2. Study design Objective Exogenous volatile organic compounds (EVOC) substrates and their bioproducts generated by the indicated enzymes have shown diagnostic potential for cirrhosis using a breath test (Table 1). We aim to generate a patent protecting these compounds for the diagnosis, triage, or any other clinical application pertinent to a diagnostic pathway, for the identification of subjects with cirrhosis of any aetiology. We aim to include also chronic liver diseases of any aetiology at pre- cirrhotic stage. Only data about cirrhosis are available for now. Table 3. List of administered substrates, enzymes involved in their metabolism, and expected bioproducts. Substrate Enzyme Bioproduct 2-Butanol αα-Alcohol 2-Butanone26dehydrogenase262-Pentanone αα-Alcohol 2-Pentanol26 / 2-methyl furan dehydrogenase26Limonene Unknown Alpha-pinene / alpha- phellandrene Nonanal Aldo-keto-reductase- 1-Nonanol27 / octane B1027 / unknown Benzyl alcohol Alcohol dehydrogenase28Benzaldehyde28Selenium Selenium cycle pathway29Dimethyl selenide29Unknown Unknown 3-Octanone Unknown Unknown 2-Chloro-m-xylene Rationale In the recent years several efficient algorithms have been developed for the detection of late- stage fibrosis and cirrhosis. However, these algorithms are based on biomarkers and diagnostic results available in secondary care, often administered to the patients only when they have developed symptoms of cirrhosis30,31when therapeutic interventions are less effective at preserving liver function. On the contrary, available methods in primary care lack adequate specificity and sensitivity for effective identification of subjects with cirrhosis before decompensation32. This short-circuits in the diagnostic pathway results in the identification of up to 75% of cirrhosis cases only after they have developed episodes of decompensation33-35. A more effective tool for early detection in primary care would allow identification of subjects earlier when causative treatments are still effective at preserving liver function and increase life expectancy35. Liver cirrhosis changes the pharmacokinetic profile of xenobiotics28,36. Compounds with high hepatic extraction, also known as flow-limited, are mainly cleared during the first pass, with a small fraction reaching systemic circulation. In the cirrhotic liver, sinusoidal capillarization and portosystemic shunt impair blood contact with hepatocytes, reducing hepatic extraction and leading to the increased bioavailability of flow-limited compounds37. On the contrary, compounds with low hepatic extraction are also known as enzyme-limited and show retarded elimination with an unchanged maximum plasma concentration and bioavailability38(Fig. 1). Given on breath concentration of exogenous volatile organic compounds (EVOCs) reflects that of the systemic circulation, breath measurement of compounds metabolized in the liver and exhaled in breath can be used for a non-invasive detection of metabolic alterations induced by the presence of cirrhosis, allowing identification of affected subjects in primary care before episodes of decompensation. The EVOCs utilized in this study exhibit varying characteristics concerning their removal by the liver and their breakdown through different metabolic pathways. Consequently, when these EVOCs are employed together in a diagnostic test, it enhances the accuracy of classification compared to using a single EVOC. Furthermore, this combined approach offers insights into alterations in liver blood flow and variations in the liver's ability to metabolize substances. A cirrhosis breath test based on this approach will provide valuable insights into expected test performance and optimum positioning of the test in a clinical pathway. Study results We present below supporting evidence from: 1. biomarker discovery and validation in baseline breath samples collected prior to any probe administration; 2. biomarker performance with EVOC approach a. model in vitro study using primary healthy human hepatocytes and isotopically labelled EVOC compounds and their potential bioproducts; b. breath analysis from clinical trial in cirrhotic patients using EVOC probe. Example 3. Breath baseline biomarker discovery and their external validation The Chile cohort is represented by subjects with cirrhosis (n = 29), or cirrhosis-free controls (n = 29) recruited in Chile38. While the UK cohort is represented by subjects with cirrhosis (n = 12), or cirrhosis-free controls (n = 12) recruited in the UK. Breath samples were collected in the morning after an overnight fasting. The Chile cohort has been used as a discovery dataset for identification of compounds that show a difference between the study groups. These compounds have been used to generate a cross-validated classification model that has been then externally validated on the UK cohort. Identified compounds showing a statistically significant difference between cirrhosis and controls in the Chile cohort (Fig. 2) were used to build several classification models and externally validated on the UK cohort. A combination of limonene with dimethyl selenide provided the best classification performance when the model was externally validated with AUC of 0.85, sensitivity of 0.75, and specificity of 0.83. This indicated that the combination of these two compounds may provide generalizable and excellent classification performance (Fig.3). Example 4. In vitro EVOCs hepatic metabolism Hepatic metabolism of limonene, 2-butanol, 2-pentanone, nonanal and benzyl alcohol was confirmed by the simultaneous treatment of human primary hepatocytes suspension with media containing 10 ng / µL of each deuterated compound. Headspace analysis using gas chromatography-mass spectrometry was used to measure the investigated compounds, and their expected bioproducts (Table 4). Control conditions omitting either primary hepatocytes or substrates were also included. Table 4. List of substrates and expected bioproducts. Substrate Bioproduct 2-Butanol-D5 2-Butanone-D5 2-Pentanone-D5 2-Pentanol, 2-methyl furan-D5 Limonene-D5 Alpha-pinene-D5, alpha-phellandrene Nonanal -D18 1-nonanol-D18, octane-D18 Benzyl alcohol-D7 Benzaldehyde-D6 2-butanol-D5, the substrate, was measured in spiked conditions, with and without hepatocytes and was absent when only hepatocytes were present (Fig. 4A). The expected bioproduct, 2- butanone-D5, was significantly more abundant when both the substrate and hepatocytes were present (Fig. 4B). These data indicate that deuterated 2-butanol and 2-butanone are not generated by primary hepatocytes per se, and that 2-butanol is converted to 2-butanone in the presence of primary hepatocytes. 2-Pentanone-D5, the substrate, was measured in spiked conditions, with and without hepatocytes and was absent when only hepatocytes were present (Fig. 5A). The expected bioproduct, 2-pentanol-D5, was significantly more abundant when both the substrate and hepatocytes were present (Fig. 5B). These data indicate that deuterated 2-pentanone and 2- pentanol are not generated by primary hepatocytes per se, and that 2-pentanone is converted to 2-pentanol in the presence of primary hepatocytes. Nonanal-D18, the substrate, was absent when only hepatocytes were present, was measured in spiked conditions without hepatocytes and was not present in the presence of hepatocytes, indicating that it was completely consumed by the hepatocytes (Fig. 6A). The expected bioproduct, 1-nonanol-D18 was significantly more abundant when both substrates and hepatocytes were present (Fig.6B). These data indicate that deuterated nonanal and 1-nonanol are not generated by primary hepatocytes per se, and that nonanal is converted to 1-nonanol in the presence of primary hepatocytes. Benzyl alcohol-D7, the substrate, was absent when only hepatocytes were present, was measured in spiked conditions without hepatocytes, was reduced in the presence of hepatocytes, and more abundant if hepatocytes were treated with 4-methilpyrazole (4-MP), an alcohol dehydrogenase (ADH) inhibitor (Fig.7A). The expected bioproduct benzaldehyde-D6 was absent when only hepatocytes were present, was reduced in spiked conditions without hepatocytes, was increased in the presence of hepatocytes, and less abundant if hepatocytes were treated with 4-MP (Fig. 7B). These data indicate that deuterated benzyl alcohol and benzaldehyde are not generated by primary hepatocytes per se, and that benzyl alcohol is converted to benzaldehyde in the presence of primary hepatocytes through the enzyme ADH. Limonene-D5 was measured in spiked conditions, with and without hepatocytes and was absent when only hepatocytes were present (Fig.8). No expected bioproduct was detected, probably due to low volatility. These data indicate that deuterated limonene is not produced by primary hepatocytes per se. Example 5. Administration of EVOCs to healthy and cirrhosis subjects Healthy and cirrhosis subjects were asked to fast overnight except water, and breath was collected before and at different time points (10, 20, 30, 40, 50, 60, 90, 120 minutes) post administration of food-grade limonene (100 mg), 2-pentanone (100 mg), 2-butanol (100 mg), nonanal (500 mg), and benzyl alcohol (500 mg). Before administration, subjects with cirrhosis have shown significantly elevated levels of limonene compared to controls (p < 0.05), while the other compounds have shown no significant differences. All the investigated compounds and their bioproducts have shown a spike in breath after ingestion (Fig.9), some compounds have shown significant different levels (p < 0.05) in breath in the time points post-administration. Table 3 summarizes the administered compounds, and the expected bioproducts, as well as the metabolic pathway generating those bioproducts. Table 5. List of administered substrates, enzymes involved in their metabolism, and expected bioproducts. Substrate Enzyme Bioproduct 2-Butanol αα-Alcohol 2-Butanone26dehydrogenase26 2-Pentanone αα-Alcohol 2-Pentanol26 / 2-methyl furan dehydrogenase26Limonene Unknown Alpha-pinene / alpha phellandrene Nonanal Aldo-keto-reductase- 1-nonanol27 / octane B1027 / unknown Benzyl alcohol Alcohol dehydrogenase28Benzaldehyde28A classification model built using the investigated compounds showed classification performance at baseline prior to the EVOCs probe administration. However, the performance was improved in the timepoints after the administration (Fig.10). Table 6. Importance of administered substrates or their hepatic bioproducts at different timepoints post-administration for classification purposes.Time Age Female BMI Octane2- 2- 2- alpha-2-pentanol Limonene nonanal 1-nonanolBenzyl ROC- butanone pentanone butanol pinene alcohol AUC 00 0 0 0 0 0 0 0 0 1.02581 0 0 0 0.7571410 0 0 0 0 -0.598044 0 1.11858 0 0 0 0 0.095333 0 0.8666720 0 0 0 0 -0.521048 0 1.16704 0 0 0 0 0.020128 -0.00573 0.8761930 0 0 0 0 0 0 0.83947 0 0.013977 0.34251 -0.11936 0 0 0.8523840 -0.0063 0 0.10919 0 0 0 0.78077 0 0.052035 0.711913 -0.427 0 0 0.8761950 0 0 0 0 0 0 0 1.19794 0.144537 0 0 0 0 0.8666760 0 0 0 0 0 0 0.3103 0.51842 0.328644 0.372775 -0.53768 -0.010985 0 0.8714390 0 0 0 0 0 0 0 0.9227 0.21179 0 -0.23823 0 0 0.84762120 0 0 0.01521 0 0 0 0 0.51484 0.78005 0 0 0 0 0.83333A lasso logistic regression was used to establish the importance of each variable for classification of subjects with cirrhosis against controls. Each row represents each investigated timepoints before (Time 0) and after ingestion of EVOCs. For each variable the coefficient is shown. Variables whose coefficient shrunk to 0 are not important for classification purposes for a specific timepoint. Limonene is the only variable contributing to classification at time 0. 2- butanone and 2-butanol were the most important variables at 10 and 20 minutes. Limonene becomes important again for classification after 30 minutes. Administration of compounds improves classification performance as indicated by the higher ROC-AUC post administration. These data indicate that administering and measuring multiple compounds improves classification performance compared to one single compound, and that the different properties of the compounds related to adsorption, hepatic clearance, and metabolism provides classification performance at different timepoints post-administration. Example 6. Methods used in LIBRA test Breath collection using the novel breath collector (NBC) (as described in GB2417686.9) A simple-to-use novel breath collector has been designed and tested. It may be handled by a patient or a nurse. A total of 100 ml of breath were collected. Sample collection is started by pushing the activation button located at the back of the device. After collection, the collector is placed in the package and shipped for sample analysis. EVOCs quantification Samples were analyzed using an Orbitrap Exploris 240 a Trace 1310 Gas chromatograph (Thermo Fisher Scientific Inc. Waltham, MA, USA) coupled with TD 100xr thermal desorption system (Markes International Ltd., Llantrisant, UK). Samples were first pre-purged for 1 min at 50 mL / min, then desorbed at 250 °C for 5 min at the same flow and focused onto a cold trap (U- T12ME-2S, Material Emissions, C4-C32 Markes International Ltd. Llantrisant, UK) at 10 °C. Analytes were then desorbed at 250 °C for 4 min into the GC column (Stabilwax-DA 30 m × 0.25 mm × 0.25 µm, Restek Corporation, Bellefonte, PA, USA) at 2 mL / min helium flow with a on the injection of 80:1. The GC temperature program was as follows: 40 °C held for 2 min, increasing to 115 °C at a rate of 15 °C / min, and to 250 at a rate of 50 °C / min and held at max temperature for 2 min. The ion mass detection was performed using electron ionization (EI) with a mass scan range from m / z 30 to m / z 450 with resolution of 60,000. EI voltage was set at 70 eV. The ion source temperature was at 270 °C. EVOCs identification and peak integration were performed with the Chromeleon Chromatography Data System (Thermo Fisher Scientific) by injecting a EVOCs standard into same type of sorbent tube or pellet and analysing them together with the breath samples (62118; Sigma Aldrich, Gillingham, UK). Limonene peak was identified using accurate m / z calculation of the main MS fragments: 93.06988, 67.05423, 79.05423, 91.05423, 121.1012. Identification criteria were: mass error < 5ppm, ion ratio < 20% based on analytical standard comparison, and retention time shift < 0.1 min. The m / z of 67.05423 was used as the quantitative mass component fragment. Areas in counts*min were used to extrapolate limonene amount in ng / 500 ml of breath by comparison with a limonene standard curve (15–2000 ng on- tube). Statistical analysis Data have been analysed using python (V.3.13.3) and R (V.4.5.0) programming languages. Multivariate statistical analysis was performed using data from all VOCs, including product / substrate ratios when available, alongside medical metadata, to define a risk model for liver cirrhosis. To build the prediction model, the LASSO regularization algorithm with 10-fold cross-validation was applied to select predictors with non-zero coefficients. This logistic regression approach penalized the absolute size of coefficients, shrinking weaker factors toward zero and reducing collinearity among VOCs. Cross-validation determined the optimal regularization parameter. Three models were developed, corresponding to baseline and 2 post-ingestion time points, featuring only the VOCs that contributed most to classification performance while minimizing the number of parameters required for testing. Predictor stability was assessed by counting selections during cross-validation; features appearing in at least 7 out of 10 folds were considered stable. Selected predictors were further analyzed in a multivariate logistic model to identify significant prognostic factors associated with cirrhosis risk. Model performance was evaluated using ROC curves, AUC, and calibration graphs, with the best-performing model selected as the final version. DeLong’s test was used to compare AUCs. A cirrhosis risk score was then constructed based on logistic regression coefficients, providing probability estimates and 95% CIs. Finally, bootstrap resampling (500 iterations) was conducted to internally validate accuracy estimates and reduce overfitting bias, ensuring better generalizability to new data. Statistical comparisons have been performed used parametric or non-parametric test based on the nature of the data distribution. A p-value < 0.05 is regarded as statistically significant. Example 7. Results from LIBRA test The following study is an ongoing multisite, cross-sectional study that aims at assessing performance of what we have termed the LIBRA test in identifying subjects with cirrhosis in a population of patients with risk factors and symptoms, including but not limited to, metabolic dysfunction-associated steatotic liver disease (MASLD), alcoholic liver disease (ALD), and viral hepatitis (VH). LIBRA relies on the ingestion of the LIBRA oral solution which contains the EVOCs limonene, 2-butanol, and 2-pentanone (100 mg each) in a formulation with high bioavailability.2-butanol and 2-pentanone are metabolized in the liver by alcohol dehydrogenase to respectively 2-butanone and 2-pentanol, all of these compounds can be measured in breath. Limonene is metabolized by CYP2C9 and CYP2C19 to more soluble compounds that are excreted in the urines. A summary is provided in Figure 11. The test procedure consists in fasting for 2 hours, ingest the LIBRA Oral Solution and breath in a collection device at different timepoints. To date a total of 171 subjects have been enrolled of which 83 have a diagnosis of cirrhosis. Presence or absence of cirrhosis has been established based on a multidisciplinary team (MDT) diagnosis routinely used in clinical practice and based on the following: 1) Physical examination to assess for presence of signs of cirrhosis. 2) Laboratory findings of blood tests. 3) Imaging modalities, or liver biopsy if available. The following results are generated from the breath samples collected. A summary of subjects characteristics by group is reported in Table 7. As expected, the cirrhosis group shows significantly higher levels of blood metrics related to liver damage. Within the cirrhosis group median levels of blood metrics usually measured in a liver function test are within the reference range, indicating a cohort of compensated, early-stage cirrhosis. Table 7. Comparison of the characteristics of cirrhosis vs controls. Continuous variables are reported as median and interquartile range [IQR]. Discrete variables are reported as number (n) and percentage (%). A classification model was built using lasso logistic regression to identify the best performing variables. The model was then refined using these variables with bootstrap resampling. The procedure was applied for all the collected timepoints (baseline - 0 min), 15 minutes, and 30 minutes). The initial variables used for the lasso logistic regression are reported in Table 8. Table 8. Variables and ratios used as a starting point to build the classification model. Baseline results Before administration of the LIBRA Oral Solution EVOCs breath levels were detectable in most of the subjects. However, in the samples where the levels were below the limit of quantification (LOQ), the value of the LOQ was used. The variables selected for baseline are reported in Table 9. Table 9. Variables selected at baseline. The selection frequency represents the fraction of times a variable was selected across the iterations. A Receiver Operating Characteristic (ROC) plot showed classification performance with AUC of 0.718 (95% CI: 0.609 - 0.819), specificity of 0.770 (95% CI: 0.581 - 0.938) and sensitivity of 0.543 (95% CI: 0.346 - 0.774). The resulting ROC plot is shown in Figure 12. Results after 15 minutes post-administration The LIBRA oral solution elicits a spike of the EVOCs in breath 15 minutes after ingestion. The variables selected for 15 minutes are reported in Table 10. Table 10. Selected features at 15 minutes. The selection frequency represents the fraction of times a variable was selected across the iterations. A ROC plot shows classification performance with an AUC of 0.819 (95% CI: 0.727 - 0.899), a specificity of 0.791 (95% CI: 0.611 - 0.938), and a sensitivity of 0.680 (95% CI: 0.471 - 0.862). A ROC plot is shown in Figure 13. Results after 30 minutes post-administration Variables selected at 30 minutes are reported in Table 11. Table 11. Selected features at 30 minutes. The selection frequency represents the fraction of times a variable was selected across the iterations. A ROC plot shows classification performance with an AUC of 0.820 (95% CI: 0.729 - 0.902), aspecificity of 0.846 (95% CI: 0.621 - 0.967), and a sensitivity of 0.627 (95% CI: 0.424 - 0.852). A ROC plot is shown in Figure 14. Box plots showing detection of EVOCs at 0-, 15- and 30-minutes post-administration to healthy subjects and subjects with cirrhosis are shown in Figures 15-17. Breath levels of each EVOC were plotted by splitting the population is cirrhosis and control group. Statistical significance was assessed using a Wilcoxon signed rank test. Conclusion The performance showed by LIBRA with an AUC > 0.8 indicates that this test can be used for identification of subjects with cirrhosis in populations bearing risk factors and symptoms.

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Claims

Claims 1. A method for detecting, diagnosing, staging, monitoring or prognosing a liver disease in a subject, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, (b) measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject.

2. A method for determining efficacy of a treatment in a subject diagnosed with a liver disease, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject before initiation of the treatment or therapy, and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a first biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, measuring the concentration of one or more endogenous bioproduct of a liver enzyme in the biological sample obtained from said subject; and (b) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject after initiation of the treatment or therapy, and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a second or subsequent biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject.

3. A method for assessing the health of a liver in advance of and / or after a surgical procedure performed in a subject, the method comprising: (a) administering two or more exogenous Volatile Organic Compounds (VOC) to the subject and measuring the concentration of the administered exogenous VOCs and / or metabolites thereof in a biological sample obtained from said subject, wherein the exogenous VOC is selected from at least one of limonene, 2-pentanone and / or 2-butanol; and, optionally, (b) measuring the concentration of one or more endogenous bioproduct of a liver enzyme in a biological sample obtained from said subject.

4. The method according to any one of claims 1-3, wherein three, four, or five exogenous VOCs are administered to the subject.

5. The method according to any preceding claim, wherein the metabolite is selected from one or more of alpha-pinene, alpha-phellandrene, 2-pentanol, 2-methyl furan and / or 2-butanone.

6. The method according to any preceding claim, wherein the exogenous VOCs are administered in combination with nonanal and / or benzyl alcohol.

7. The method according to claim 6, wherein the metabolite is further selected from 1-nonanol, octane, and / or benzaldehyde.

8. The method according to any preceding claim, wherein the endogenous bioproduct is dimethyl selenide.

9. The method according to any preceding claim, wherein the biological sample obtained from a subject is obtained prior to the administration of exogenous VOCs, and at multiple time points post administration.

10. The method according to claim 9, wherein the biological sample obtained from the subject is obtained between 0 and 24 hours post administration of exogenous VOCs.

11. The method according to any preceding claim, wherein the method further comprises comparing the concentration of the compounds measured in the biological sample obtained from the subject to the concentration of the same compounds measured in a biological sample obtained from a healthy subject, wherein a difference in the concentration of measured compounds, the ratio between substrate and bioproduct, the ratio between administered VOCs, the ratio between different metabolites and / or ratio with a standard compound indicates a likelihood of a liver disease.

12. The method according to claim 11, wherein the concentration of the measured compounds in the biological sample obtained from the test subject are elevated or reduced compared to the concentration of said compounds in a biological sample obtained from a healthy subject.

13. The method according to claim 11, wherein the ratio between substrate and bioproduct is measured, wherein the substrate is selected from one of limonene, 2-pentanone and, 2-butanol and the bioproduct is selected from one of alpha-pinene, alpha-phellandrene, 2-pentanol, 2- methyl furan and / or 2-butanone.

14. The method according to any preceding claim, wherein the method further comprises comparing the ratio between the concentration of an administered VOC and a physiological characteristic of the subject to the ratio between the concentration of the same administered VOC and a physiological characteristic of a healthy subject, wherein a difference in the ratio between the concentration of an administered VOC and a physiological characteristic indicates a likelihood of a liver disease.

15. The method according to claim 11, wherein the administered VOC is selected from one or more of limonene, 2-pentnaone and / or 2-butanol, and the physiological characteristic is selected from one or more of the subject’s Body Mass Index, Body Surface Area, height, weight, sex, age and / or blood volume.

16. The method according to any preceding claim, wherein the biological sample is selected from breath, urine, blood, serum, and / or tissue.

17. The method according to claim 16, wherein the biological sample is exhaled breath.

18. The method according to any preceding claim, wherein the exogenous VOCs are administered at a dose of between 0.1 to 1000 mg.

19. The method according to any preceding claim, wherein the administered exogenous VOCs are administered simultaneously, sequentially or separately.

20. The method according to any preceding claim, wherein the administered exogenous VOC is formulated as a liquid, a tablet or capsule for fast release.

21. The method according to any preceding claim, wherein the administration of the exogenous VOCs is after a fasting period.

22. The method according to claim 21, wherein the subject has fasted for 1-12 hours.

23. The method according to any preceding claim, wherein the administration of exogenous VOCs is oral or parenteral.

24. The method according to any preceding claim, wherein the liver disease is a liver disease associated with cirrhosis, portal hypertension, liver fibrosis, metabolic dysfunction-associated steatotic liver disease (MASLD), alcoholic liver disease (ALD), viral hepatitis, alcoholic hepatitis, autoimmune hepatitis, Wilson’s disease, alpha-1 antitrypsin deficiency, drug-induced liver injury,acetaminophen toxicity, hepatic encephalopathy, Budd-Chiari syndrome, primary biliary cholangitis, primary sclerosing cholangitis, and / or haemochromatosis.

25. The method according to claim 24, wherein the cirrhosis is compensated or decompensated.

26. The method according to any preceding claim, wherein the exogenous VOC, metabolite and / or endogenous bioproduct is labelled.

27. The method according to claim 26, wherein the label is deuterium.

28. The method according to any preceding claim, wherein the concentration of compounds is measured by spectrometry.

29. The method according to claim 28, wherein the spectrometry is gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry.

30. A method for determining liver function comprising a method according to any of claims 1 to 29.

31. A method for triaging a subject with liver disease comprising a method according to any of claims 1 to 29.

32. A method for identifying a subject with cirrhosis suitable for causative treatments comprising a method according to any of claims 1 to 29.

33. The method according to claim 32, wherein the causative treatment is selected from viral suppression / eradication, alcohol abstinence, lifestyle improvements, and / or early treatment of precipitating risk factors including dehydration, hypotension, and / or infections.

34. A method for identifying a subject suitable for advanced management treatment comprising a method according to any of claims 1 to 29.

35. The method according to claim 34, wherein the advanced management treatment is selected from fluid resuscitation, vasopressors, endoscopic band ligation, injection sclerotherapy, trans- jugular intrahepatic portosystemic shunt (TIPS), paracentesis, lactulose and / or liver transplant.

36. A method indicating the likelihood of liver cirrhosis according to any of claims 1 to 29.

37. The method according to claim 36, wherein the method further comprises the generation of a risk score based on the combination of quantitative measurements of exogenous VOCs and / or endogenous bioproducts from an exhaled breath sample.

38. The method according to claim 37, wherein the quantitative measurements of exogenous VOCs and / or endogenous bioproducts from an exhaled breath sample is combined with demographic and / or clinical metrics to generate the risk score.

39. A composition comprising one or more exogenous VOC, wherein the VOC is selected from limonene, 2-pentanone and / or 2-butanol.

40. The composition of claim 39, wherein the composition additionally contains nonanal and / or benzyl alcohol.

41. A kit comprising the composition according to any of claims 39 or 40, and a device for capturing a breath sample from a subject.

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