Assay for monitoring crohn's disease and mitochondrial dysfunction

Abstract

The present application provides an ultrasensitive colorimetric assay as well as an early biomarker for patient monitoring and medical treatment of patients suffering from a possible mitochondrial dysfunction, inflammatory bowel disease, particularly Crohn's disease. The ultrasensitive colorimetric assay measures the level of L-citrulline in a plasma or serum sample; and when the level of L-citrulline in plasma or serum decreases or falls even below 30 µmol L-citrulline, this indicates a mitochondrial cell disorder caused by a relapse or an increase in intestinal inflammation due to a flare of Crohn's disease. A method of detecting and treating the mitochondrial dysfunction is also provided.

Description

ASSAY FOR MONITORING CROHN'S DISEASE AND MITOCHONDRIAL DYSFUNCTIONFIELD OF THE INVENTION

[0001] The present invention relates to a colorimetric assay of citrulline, particularly a colorimetric assay for monitoring Crohn’s disease, mitochondrial dysfunction and concurrent inflammations.DETAILED DESCRIPTION OF THE INVENTION

[0002] The diagnosis of inflammatory bowel disease (IBD) includes conditions that result from inflammation of the digestive tract, causing abdominal pain and symptoms. Crohn's disease (CD) is a form of IBD that can affect any part of the gastrointestinal tract, with the terminal ileum of the small intestine being the most affected area. Symptoms include abdominal pain, diarrhoea, fever, weight loss, an increased risk of colon and small bowel cancer, and, in chronic cases, bowel obstruction. Due to the location of the inflammation, Crohn's disease can lead to vitamin deficiencies, including vitamin B12, folic acid, and iron deficiency anaemia., Inflammation in the duodenum and jejunum can also affect the absorption of other nutrients, and if the stomach is affected, intrinsic factor production may be reduced. In addition, the disease can lead to an increased risk of gallstones due to reduced resorption of bile acid in the ileum and excretion of bile in the faeces.

[0003] Crohn's disease can affect more than just the gastrointestinal tract. It is also associated with neurological complications (reported in up to 15%) such as seizures, stroke, and depression, or a rheumatological condition called seronegative spondyloarthropathy, which causes inflammation of the joints and / or muscle attachments (enthesitis), leading to symptoms such as joint pain, stiffness, and reduced mobility. They can affect large weightbearing joints (hip, knee) and small joints in the hands and feet, and even the spine, leading to ankylosing spondylitis.

[0004] Crohn's disease can also affect the blood, endocrine system, and skin. It increases the risk of blood clots and autoimmune haemolytic anaemia, which can cause fatigue and a pale appearance. Common skin manifestations include erythema nodosum, which appears as raised, tender red nodules, and pyoderma gangrenosum, which is typically a painful ulcerative nodule. Additionally, Crohn’s disease also increases the risk of osteoporosis, or thinning of the bones, or can cause clubbing, a deformity of the fingertips. Crohn’s disease can also cause inflammation of the uvea (the inner portion of the eye), leading to blurred vision, eye pain, and if left untreated, vision loss.

[0005] The cause of Crohn's disease is unknown, but genetics play a role. It is a chronic disease in which the body's immune system attacks the gastrointestinal tract, possibly targeting microbial antigens. Although Crohn's disease is immune-related, it is not an autoimmune disease. Smokers are more likely to develop Crohn's disease than non-smokers. It is usually diagnosed in the teens and twenties but can occur at any age. Symptoms of Crohn’s disease often can often go undiagnosed for years before a diagnosis of the disease is made. There is currently no cure for the disease, and treatment options focus on managing the symptoms, preventing flares, and maintaining remission. A corticosteroid may be used for a short time to quickly improve symptoms, along with another medication such as methotrexate or thiopurine. Half of people with the disease will need surgery at some point in the next ten years. The state of the art represents a problem, and there is an urgent need for a method and an in-vitro test to enable the monitoring of patients suffering from Morbus Crohn.BRIEF DESCRIPTION OF THE INVENTION

[0006] The present disclosure provides a method for monitoring patients suffering from Inflammatory Bowel Disease, in particular Morbus Crohn, characterised by the steps: -(i) obtaining an in-vitro sample of plasma or serum from the patient;(ii) measuring the concentration of L-citrulline in said plasma or serum sample; and(iii) comparing the concentration of L-citrulline found in the plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline is indicative of increased mitochondrial dysfunction preceding or accompanying a relapse or increase in Crohn’s disease.

[0007] In an embodiment of the invention, a measured L-citrulline concentration below 30 pmol / L in the patient’s plasma or serum is indicative of mitochondrial dysfunction and inflammation. A decreased L-citrulline concentration below 30 pmol / L in the patient’s plasma or serum is indicative of a deficiency and a need to support mitochondrial function by providing the patient with an alternative energy source, additional antioxidants, nutritional supplements and vitamins.

[0008] In a preferred embodiment of the invention, a measured L-citrulline concentration below 30 pmol in the plasma or serum of a patient suffering from Morbus Crohn indicates a relapse or increased inflammation due to mitochondrial cell disorder.

[0009] In some embodiments, the method of monitoring patients from Morbus Crohn comprises the use of tandem mass spectrometry, amino acid profiling, and / or immunoassay for determining the L-citrulline level in a patient’s plasma or serum.

[0010] In some embodiments, the method of monitoring patients suffering from Morbus Crohn comprises a colour reaction for determining the L-citru Hine level in a patient’s plasma or serum.

[0011] In some embodiments, the method of monitoring patients suffering from Morbus Crohn comprises subjecting a patient’s plasma or serum sample to an enzymatic treatment with urease prior to the colour reaction for determining the L-citrulline level.

[0012] In some embodiments, the method of monitoring patients suffering from Morbus Crohn comprises subjecting a patient’s plasma or serum sample to an enzymatic treatment with urease and to a deproteination treatment prior to the colour reaction for determining the L-citrulline level.

[0013] A preferred aspect of the invention relates to a method of monitoring patients suffering from Morbus Crohn or inflammatory bowel disease comprising the steps of: -(i) obtaining an in-vitro sample of plasma or serum taken from a patient;(ii) subjecting the sample to enzymatic treatment with urease to render it substantially urea free;(iii) subjecting the sample to a deproteination treatment to obtain a substantially protein- free liquid sample;(iv) sequentially adding (A) an aqueous solution containing diacetyl monoxime (DAM) and thiosemicarbazide in acetic acid, and (B) an acidic solution containing a catalytically active iron or manganese redox agent; and(v) heating the sample mixture to above 70 degrees Celsius to form chromophores having a peak absorbance in the range of 530 to 560 nm; and(vi) measuring the colour intensity at 540 nm in comparison with a series of L-citrulline standards in a salt solution to determine the concentration of L-citrulline in the plasma or serum sample; and(vi) comparing the concentration of L-citrulline found in said plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline indicates relapse or an increase in Crohn’s disease.

[0014] In some embodiments, solution B contains 3 M phosphoric acid, 6 M H2SO4, and 0.1 to 5 mM NH4Fe(SO4)2, preferably 2 mM NH4Fe(SO4)2.

[0015] In some preferred embodiments, solution A contains diacetyl monoxime and thiosemicarbazide in a molar ratio of 30 to 70, preferably 40 to 50.

[0016] In some embodiments, solution A and solution B are mixed in a ratio of 1 :3.

[0017] In some embodiments, the sample with the colour development solution is heated to 70 to 100 degrees Celsius for 10 to 60 minutes, preferably 10 to 20 minutes.

[0018] In some embodiments, the chromophores formed in the assay are substituted triazine chromophores having a peak absorbance in the range of 530 to 540 nm.

[0019] In some embodiments, the volume of the plasma or serum sample to the volumes of the urease solution and the trichloroacetic acid solution is greater than 20:1 and 10:1 , respectively, to increase the sensitivity and lower the measurement limit of the photometric assay.

[0020] In some preferred embodiments, the lower measurement limit of the photometric assay is 1 ng L-citrulline (0,005 pmol).

[0021] Another aspect of the invention is a kit of parts for photometric testing and monitoring of patients suffering from inflammatory bowel disease, in particular from Morbus Crohn, the determination of L-citrulline in a plasma or serum sample comprising :- an aqueous urease concentrate for treating and removing any substantial urea from the plasma or serum sample without diluting the sample by more than 5 percent, a concentrate solution of trichloroacetic acid for deproteinization of the sample without diluting the sample by more than 10 percent, a solution (A), containing diacetyl monoxime and thiosemicarbazide, and an acidic solution (B) containing a catalytically active iron- or manganese-containing redox agent suitable for the synthesis of triazine chromophores of L-citrulline having a peak absorption in the range of 530 to 560 nm, and one or more aqueous standard solutions of L-citrulline, suitable for the photometric quantitative determination of L-citrulline in plasma or serum in the concentration range from 0.5 to 100 pmol / L, in order to allow, by a photometric assay, the monitoring of flares and relapses of Crohn's disease in a patient.

[0022] Further embodiments and advantages of the invention will become apparent from the detailed description and drawings.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In the drawings: -Fig. 1. is a graph comparing plasma levels of L-citrulline and heat shock protein 60 in healthy subjects and patients with Crohn’s disease.Fig. 2 is a graph comparing plasma levels of L-citrulline and TNFa in healthy subjects and patients with Crohn’s disease.DETAILED DESCRIPTION OF THE INVENTION

[0024] Several citrulline tests are known in standard clinical practice. They are not used to supervise the treatment and recurrence of Crohn's disease. More specifically, EP4032969 discloses a method for quantifying citrulline in a sample by adding an enzyme using citrulline as the substrate. This enzyme can be a citrulline oxidoreductase. The addition of citrulline oxidase produces hydrogen peroxide, which can be quantified by standard methods or an electrochemical sensor. Alternatively, citrulline dehydrogenase can be added and the citrulline concentration determined by reducing a mediator. An electrochemical citrulline sensor is also provided to use citrulline as a biomarker of diseases associated with abnormal citrullination of proteins, such as multiple sclerosis, Alzheimer's disease, chronic rheumatoid arthritis, psoriasis, prion disease, liver fibrosis, chronic obstructive pulmonary disease, cancer, and the like. In contrast to these methods, the method of the invention discloses a measurement of free citrulline in plasma or serum for monitoring Crohn’s disease. In addition, indirect enzymatic measurements are not sensitive enough and are susceptible to solution pH, interference from divalent cation, and incomplete enzymatic reactions.

[0025] Citrulline concentration in plasma or serum is also used as a surrogate marker for nitric oxide (NO) production. Firstly, nitric oxide is a well-known intra- and intercellular signalling molecule. It is produced in the vascular endothelium in cases of vascular dysfunction. This nitric oxide diffuses into the neighbouring vascular smooth muscle cells (VSMCs). There it activates the enzyme soluble guanylate cyclase (sGC), which increases intracellular cGMP production from GTP. This leads to relaxation of the vascular smooth muscle cells and vasodilatation. Endothelial dysfunction in coronary artery disease is also a critical factor in vasospasm, ischaemia, and thrombosis. Secondly, citrulline is synthesised in the body in the citrulline-NO-cycle by NO-synthase-catalysed oxidation of arginine. This reaction produces citrulline and nitric oxide in a product stoichiometry of 1 :1. The conversion of arginine to citrulline is, therefore, proportional to NO synthesis, and the increased citrulline concentration in plasma or serum is a measure of the severity of endothelial or coronary artery disease as well as for some specific dermatologic autoimmune diseases (Wanchu A et al., Serum and Urine Nitrite and Citrulline Levels among Patients with Systemic Lupus Erythematosus: A Possible Addition to Activity Parameters?, J Clin Rheumatology, Practical Reports on Rheumatic & Musculoskeletal Diseases 2001 Feb. 7 (1 ), 10-5). The degree of these diseases or the seriousness of these disease states is therefore regularly associated with elevated citrulline levels in the plasma or serum of these patients. These known correlations do not establish a causal relationship between plasma or serum citrulline levels and Crohn's disease (CD) or IBD.

[0026] Independently, the present inventors have discovered that plasma or serum L- citrulline levels tend to be low in Crohn's disease and that a decrease in plasma L -citrullinelevels regularly precedes relapse and recurrence of Crohn's disease symptoms. To use this discovery as a diagnostic tool in the treatment of Morbus Crohn, it is, therefore, necessary to enable clinical laboratories to measure L-citrulline in plasma or serum at extremely low concentrations in the range of 0.5 to 30 pmol / L. In a photometric citrulline assay, this can only be achieved by removing any urea present in the plasma or serum and performing the colour reaction with only minimal dilution of the plasma or serum sample. This is because the colour reaction of diacetyl monoxime and citrulline is as follows:Diacetyl monoxime + citrulline triazine chromophores with 530 - 560 nm.

[0028] Such condensation with diacetyl monoxime is also used in the Fearon reaction to determine urea concentration in urine, plasma, and serum. In other words, removing any urea present in the sample is of utmost importance as diacetyl monoxime reacts with urea to form an interfering chromophore. The urea present in plasma or serum is an end-product of the body and is formed in the liver from ammonia produced by the deamination of amino acids. In medical diagnostics, the terms urea and blood urea nitrogen (BUN) are used interchangeably. Urea, together with creatinine, is a biomarker for the differential diagnosis of chronic renal insufficiency and acute renal failure, as the kidneys excrete it and are, therefore, a marker of renal function and a biomarker of the patient's metabolic status. The range of serum or plasma urea is usually in the range from 1.5 to 9.5 mmol / L (10 to 60 mg / dL), depending on age, sex, and laboratory, which is approximately 1000 times the molar concentration of citrulline in plasma. Therefore, urea in the blood may interfere with a photometric measurement of citrulline.

[0029] On the other hand, there is a connection between citrulline and the urea cycle, which arises from the fact that citrulline is enzymatically produced in the body from ornithine. Ornithine is an intermediate in the urea cycle. A urea cycle disorder affects all biochemical processes in which waste nitrogen and ammonia are converted into urea. The symptoms of a urea cycle disorder vary in severity, but all result from excessive accumulation of ammonia in the blood and body tissues. Typical symptoms include loss of appetite, vomiting, drowsiness, seizures, protein intolerance, and coma. In addition to citrullinemia, the urea cycle disordersinclude carbamoyl phosphate synthetase (CPS) deficiency, argininosuccinic acid lyase deficiency, ornithine transcarbamylase (OTC) deficiency, arginase deficiency, and N-acetyl glutamate synthetase (NAGS) deficiency.

[0030] However, the ornithine transcarbamylase (OTC), which is required for the conversion of ornithine to citrulline, is remarkably inhibited when the mitochondrial unfolded protein response is activated. This, in turn, leads to a decrease in plasma or serum citrulline levels. Without wishing to be bound by theory, the inventors have found in ileal tissue samples from Crohn’s patients and by comparison with mouse models for mitochondrial dysfunction (hsp60A / Aisc) and Crohn’s disease-like ileitis (TNFAARE) that reduced Paneth cell function in ileal tissue correlates with mitochondrial dysfunction. The degree of mitochondrial dysfunction can be assessed by increased levels of the chaperone hspQO and decreased numbers of Lysozyme-positive granules in the cytoplasm of Paneth cells; see Khaloian S et al. in Mitochondrial impairment drives intestinal stem cell transition into dysfunctional Paneth cells predicting Crohn's disease recurrence, Gut 2020 (1 1 ), 1939-1951 . The mitochondrial heatshock protein hsp60 is required for the assembly into oligomeric complexes of proteins imported into the mitochondrial matrix, and its increase is a sign of a structurally impaired mitochondrion. On the other hand, it has been observed that inflammation in the mouse model of Crohn’s-like ileitis is associated with both mitochondrial dysfunctions, leading to a reduced number of cytoplasmic granules in Paneth cells, and a histological morphology like that seen in ileal samples from Crohn’s patients. This was confirmed histologically in tissue samples from Crohn’s patients who underwent resection surgery, where the number of Paneth cells and granular Paneth cells was significantly reduced in tissues classified as inflamed at the time of surgery.

[0031] Crohn's disease (CD) is a pathological condition characterised by acute and chronic inflammation of the intestinal mucosa and terminal ileum. Various genetic risk factors have been described for the dysregulated immune response to the commensal microbiota, which results in reduced numbers of goblet cells and Paneth cells that participate in the mucosal defence, pathogen clearance, and shaping of the commensal microbiota. Reduced Paneth cell function appears to correlate with microbiota-driven Crohn’s disease and ileal inflammation. However, a major challenge in the treatment of Crohn’s disease is the heterogeneity of the disease, with only subsets of patients responding to therapies such as anti-TNF antibodies. The number of genes involved in Crohn’s disease, the range of dietary and environmental triggers, and bacterial infections imply diverse disease ainsulin resistance and is associated with obesity-induced type 2 diabetes. As a cytokine, TNFa is used by the immune system for cell signalling. If macrophages detect an infection, they release TNFa to alert other immune system cells as part of an inflammatory response. Dysregulation of TNFa production has been implicated in a number of human diseases. Although controversial, somestudies have even linked depression and IBD to increased levels of TNFa. Reduced plasma or serum citrulline levels following a urea cycle disorder or mitochondrial dysfunction can be associated with numerous symptoms.

[0032] Generally, the heat shock protein 60 (HSP60) is a marker for assessing mitochondrial function. Studies conducted in yeast have demonstrated that HSP60 plays a critical role in mitochondrial protein homeostasis. In humans, mutations in the hsp 60 gene can result in a severe neurodegenerative disorder known as hereditary spastic paraplegia, likely caused by mitochondrial dysfunction. A deficiency of HSP60 deficiency can affect the viability of cells, potentially leading to embryonic lethality. Conversely, elevated HSP60 expression is associated with an activated MT-UPR (mitochondrial unfolded protein repression) signalling pathway linked to unfolded proteins in the epithelial cells of individuals with IBD. Nonetheless, the mechanisms of the MT-UPR signalling are not well understood. To investigate the role of the mitochondrial chaperone HSP60 in regulating epithelial cell homeostasis in the intestine, epithelial-specific HSP60 knockout mice were generated and studied. These studies revealed that HSP60 deficiency causes mitochondrial dysfunction and disrupts protein homeostasis in epithelial stem cells. The malfunctioning mitochondria release signals related to wnt (a crucial signalling pathway for cell growth and development), leading to excessive proliferation of the remaining stem cells unaffected by the hsp60 gene knock-out.

[0033] The layer of cells in the intestine, known as the intestinal epithelial cell layer, is vital for maintaining a balanced and healthy gut. It acts as a barrier between the internal environment of the body and the outside world, playing a key role in intestinal homeostasis. When this balance is disrupted, it can lead to chronic degenerative diseases like colorectal cancer (CRC) and inflammatory bowel diseases such as Crohn's disease (CD). Within the intestinal epithelium, stem cells in structures called crypts give rise to different types of cells. Through cycles of division and growth, stem cell-derived progenitor cells develop into absorptive enterocytes or various secretory cells (goblet, enteroendocrine, and tuft cells). Paneth cells, on the other hand, descend directly from stem cells and remain within the crypt to contribute to antimicrobial defence and stem cell maintenance. Problems in maintaining the balance of epithelial cells can result in defects in antimicrobial defence, barrier function, and the interaction between intestinal epithelial cells (lECs) and immune cells. These issues play a crucial role in the development of IBD and CD. Furthermore, chronic inflammation significantly increases the risk of CRC, particularly in individuals with IBD or CD. Many of the genetic factors identified so far in relation to CRC are associated with the regulation of cell proliferation.

[0034] To maintain a balanced and functional state of intestinal epithelial cells (IEC), it is crucial to regulate and adjust the abundance and capacity of organelles in response to the actual cellular demand. One key factor that limits the function of organelles and cells is theavailability of properly folded and functional proteins. Unfolded protein responses are auto regulatory mechanisms that evolved in the cytoplasm, the endoplasmic reticulum (ER), and mitochondria to ensure adaptation to varying demands for proteins within cells. Triggers for disruptions in protein homeostasis include infections, oxidative stress, and metabolic alterations. Unfolded protein repression in the endoplasmic reticulum is critical for adequately functioning Paneth and Goblet cells, as these cells are specialised in the production and secretion of proteins assembled in the ER.

[0035] As previously discussed, disorders affecting mitochondrial function and homeostasis result in decreased citrulline levels in plasma and serum. Consequently, the measurement of citrulline in plasma or serum serves as a valuable biomarker for assessing mitochondrial function, mitochondrial unfolded protein repression, and protein homeostasis. This is especially relevant in intestinal epithelial cells where infections, oxidative stress, intestinal microbiota, and metabolic changes can influence such. This established a pressing need to accurately monitor and measure low and extremely low concentrations of citrulline in plasma and serum, necessitating an improved measurement protocol when using a conventional photometric assay to determine citrulline. This is particularly crucial for individuals with IBD or Morbus Crohn, as a decrease in citrulline levels in plasma or serum precedes a relapse and flare-ups of Crohn’s disease.

[0036] Diacetyl monoxime is one of the more stable reagents. It is the focus of this photometric assay due to its fast reaction with conjugated diamines, as present in citrulline, and the resulting highly red chromophore intensity and stability (Knipp, M & Vasak M, A Colorimetric 96-Well Microtiter Plate Assay for the Determination of Enzymatically Formed Citrulline, Anal Biochem 2000, 286, 257-64. doi:10.1006 / abio.2000.4805). However, the Fearon reaction with urea is a problem. This problem is solved by treating the sample with highly active concentrated urease in acidic buffer to avoid dilution of the sample and lowering the detection limit for citrulline. A problem that did not bother in the prior art because it focused on increased citrulline concentrations in plasma, serum, or urine. Condensation of conjugated amines with diacetyl monoxime has been extensively studied to improve the reproducibility and linearity of the response. Still, the additional problem is that low concentrations or the additional dilution of the reaction mixture leads to a deviation from Beer’s law, and the chromophore response curve no longer passes through the origin. This problem has been solved by optimising the citrulline colour reaction by performing a prior urease reaction in an acidic environment and the addition of a redox catalyst so that the condensation reaction to produce highly intense chromophores with citrulline works at low and extremely low concentrations of citrulline in the sample. Therefore, the analysis of a sample in duplicate takes less than 1 hour.

[0037] According to the invention, the colour reaction of L-citrulline with diacetyl monoxime is preferably carried out in 3 M phosphoric acid and 6 M sulphuric acid at 90 to 95°Cfor 5 to 60 minutes, preferably 10 to 15 minutes, in the presence of ferric ammonium sulphate (ammonium iron (III) sulphate) instead of a ferrous iron (II) salt as redox catalyst. These improved conditions result in an intensely red chromophore, which follows the Beer-Lambert law so that the absorbance at 530-540 nm is proportional to the concentration of L-citrulline even at low concentrations. The chemical reaction is preferably carried out in the wells of a 96- well polystyrene microtiter plate. The method is rapid, requires little sample material and can be performed in a volume of less than 250 pL. The detection limit for L-citrulline is as low as about 10 ng (about 0,05 nmol) so that plasma concentrations of L-citrulline below 30 pmol can easily be measured and monitored in a photometer.EXAMPLE 1

[0038] Plasma samples were obtained in intervals from a patient with Crohn’s disease and plasma L-citrulline concentration was monitored together with HSP60, wnt and TNFa which are markers for systemic inflammation and, as shown here, for mitochondrial dysfunction.

[0039] TNFa is a cytokine secreted by immune cells, such as macrophages, monocytes, neutrophils, T-cells, and natural killer cells. Its diverse biological functions, including triggering the acute phase reaction and promoting cellular proliferation and differentiation. Increased TNFa levels are generally found in patients with Crohn’s disease, ulcerating colitis, or rheumatoid arthritis. The IDK® TNFa ELISA kit (Immundiagnostik AG, Bensheim, DE, Art. No. K9610) was used to measure TNFa levels in plasma.

[0040] The human 60-kDa heat-shock protein (hsp60) is a key target for T cell and antibody response during chronic inflammation. The proinflammatory macrophage response is hsp60 dose-dependent and resembles the effects of lipopolysaccharide stimulation in terms of kinetics and magnitude. HSP60 is also a recognized danger signal for the innate immune system. The HSP60 level was determined in plasma using a commercial HSP60 ELISA kit (Invitrogen, Cat. No. EH244 RB).

[0041] De-regulation and abnormal levels of wnt molecules are associated with various human diseases, including cancer and degenerative disorders. Wnts have a crucial role in regulating tissue cell behaviour and differentiation, and wnt / / 3-catenin signalling pathway strongly influences cell fate along the crypt-villus axis, wnts were determined in plasma using a commercial human wnt3A ELISA kit (Biomatik Corp., Cat. No. 50-149-9636).

[0042] In this representative example, the L-citrulline in human plasma was measured using the herein described modified protocol of an L-citrulline kit (Immundiagnostik AG, Bensheim, Cat. No. K6600) to increase its measurement sensitivity and further lower the limit of citrulline detection. This was achieved by treating 500 pl of the plasma sample with 25 pl offreshly prepared concentrated urease (50 kU / ml) from Canavalia ensiformis in 50 mM acetate buffer at pH 6.0, at 37°C for 1 hour. This treatment helped to remove absolutely all of the urea present in the plasma and to reduce the background of the colorimetric assay. The plasma proteins were then precipitated by the addition of 40 pl of trichloroacetic acid followed by colling the mixture in an ice bath for 30 minutes. After centrifugation at 3000 g for 10 minutes, 100 pl of the pre-treated urea-free sample was combined with 150 pl of freshly prepared ultrasensitive colour development reagent as described below.

[0043] The ultrasensitive color development reagent was prepared by freshly mixing 1 volume of solution A with 2.5 volumes of solution B only. Solution A was prepared by dissolving 1 .62 mg diacetyl monoxime (80 mM DAMO) and 36 mg thiosemicarbazide (TSC) in 200 ml of H2O. This solution was stored at 4°C in the dark until use. Solution B contained 3 M H3PO4, 6 M H2SO4, and 2 mM NH4Fe(SC>4)2. The rationale behind this mixture is to protect the developing red dye from NH2OH, a by-product of the reaction between citrulline and diacetyl monoxime. The thiosemicarbazide acts as a reducing agent in this mixture and is required for rapid colour formation. It also shifts the peak absorbance of the developing red dye to between 530 and 540 nm, where there is less interference from various protein cofactors, urea, and other additives. However, the reaction of the conjugated carbamidates group in citrulline and diacetyl monoxime only occurs in a strongly acidic environment and requires the addition of an Fe(lll) redox catalyst which is essential for intense color formation. Taken together, these modifications increase the sensitivity of this colorimetric assay to allow the measurement of L- citrulline down to 10 ng (0,05 nmol) and below, and the determination of L -citrulline in plasma or serum in the range below 30 pmol, which is critical for the clinical detection of mitochondrial dysfunction caused by the developing inflammatory cascade in Crohn’s disease.

[0044] The standard calibration curve was prepared as follows: A stock solution of L- citrulline (40 mmol / L) was diluted 1 :100 in 50 mM phosphate buffer, pH 6.5 (STDBUF), to obtain Standard 1 (400 pmol L.-Cit / L). This standard was then serially diluted 1 :2 in six steps with phosphate buffer (STDBUF) to obtain Standard 2 (200 pmol / L); Standard 3 (100 pmol / L), Standard 4 (50 pmol / L); Standard 5 (25 pmol / L), Standard 6 (12.5 pmol / L), Standard 7 (6.25 pmol / L). The blank dilution buffer (STDBUF) was Standard 8 (0 pmol L-Cit. / L). Because the colour development reagent itself develops a yellowish colour at 95°C, it is important to establish its spectral contribution (blank). The corresponding difference absorption spectrum of the chromophore reveals a broad spectral envelope with an absorbance peak at 530 - 540 nm at which the contribution of the blank is rather low. In this assay, the widely available 540 nm filter can be used as at this wavelength the reduction of sensitivity compared to the less common 530 nm filter is less than 9%.

[0045] The colour reaction was parallelly performed in the wells of a 96-well polystyrene microtiter plate. The microtiter plate was sealed with parafilm® to avoid sampleevaporation, placed onto a preheated aluminum frame and incubated at 95°C for 15 minutes in the oven. The microtiter plate was then allowed to cool at room temperature for 10 minutes. The red colour development in the samples was measured using a microtiter plate reader at 540 nm (DYNAREAD, Dynex Technologies, Praha, CZ). A rapid dye formation requires an increased temperature (95°C) but maximum absorbance is reached after 15 min of the development time. After the heating, the samples were allowed to cool down to room temperature for approx. 10 min to complete the colour-developing process. Since the developed light-sensitive dye is stable for at least 20 min, the absorption measurements were performed within one hour.

[0046] The results are summarized in Figures 1 and 2. Figure 1 is a bar graph comparing plasma levels of L-citrulline and heat shock protein 60 in healthy volunteers (Ctrl) and patients with diagnosed Crohn's disease (D2). Fig. 2 compares in a bar graph the plasma levels of L-citrulline and wnt in medicated Crohn's disease patients, once without significant inflammatory events and once with the onset of increased inflammatory events due to a Crohn's disease flare. The bar graphs also show that the 30 pmol plasma cut-off is a useful early biomarker for monitoring and medication management of Crohn's disease patients.EXAMPLE 2

[0047] To validate the L-citrulline measurements, samples from 9 healthy volunteers, including 6 females and 3 males, were measured, and it was also investigated whether the L- citrulline level was condition dependent or changed in the fasting state or in the case of haemolysis of the sample (in two different samples).

[0048] The first patient cohort included samples (M0) from 64 non-active and 33 active Crohn's disease (CD) patients undergoing resection surgery. Samples were from a prospective multicentre study performed by the REMIND group (see Auzolle C et aL, Male gender, active smoking and previous intestinal resection are risk factors for postoperative endoscopic recurrence in Crohn's disease: results from a prospective cohort study. Aliment Pharmacol Ther. 2018, 48(9), 924-932). Endoscopic recurrence of the patients was assessed based on a postoperative endoscopy that was performed 6-12 months after surgery (M6) and was defined as a Rutgeerts score. Patients with a Rutgeerts score of < 2 were categorised as non-recurrent, whereas a Rutgeerts score of >2 indicated clinical recurrence.

[0049] The second patient cohort included 168 serum samples from 52 non-inflamed (clinical GvH scale 0-1 ) and 27 inflamed (clinical GvH scale 2-4) GvH disease patients. Additionally, for 13 patients, we got access to at least seven serum samples collected at different time points post-transplantation from each patient. GvH disease patients experiencedinflammation either in the upper Gl, including the stomach, the duodenum, and the terminal ileum or in the lower Gl, including the colon.

[0050] The third cohort included serum samples from 6 patients at five different time points (before transplantation, at dO, d7, and d14 after transplantation and GvHD samples).

[0051] The citrulline assays in this study were performed as described in Example 1 but adapted to the available sample size. 55pl of a serum sample from each patient was added to 5|_il ddH2O and mixed well. 12pl of reconstituted concentrated urease was added to the sample, mixed, and incubated at 37°C for 1 h. To precipitate proteins and cofactors, 18pl of cold TCA reagent was added to the sample and mixed well. Samples were incubated at 4°C for 30 min, centrifuged at 3000g for 10 min, and the supernatant was separated. These steps were performed to eliminate the binding of other substances. For the assay procedure, 40pl of the supernatant from each sample was pipetted into the microtiter plate (MTP). 133pl of colour solution (mixture of 1 x solution A and 3x solution B) was added to each sample. The microtiter plate was covered and incubated in a preheated incubator at 90°C for 15 min. The citrulline concentration was measured at 540nm using a MULTISCAN spectrometer (Thermo Labsystem). These experiments were done to confirm the results of the representative example 1 and to investigate whether a suppression of mitochondrial dysfunction can prevent a flare of Crohn’s disease.

[0052] The treatment of mitochondrial dysfunction can be challenging due to the complexity of this disorder and its many aetiologies. There are several drugs and therapeutic approaches that are used to manage symptoms and improve the quality of life for patients. This disclosure focuses on supporting mitochondrial function, protecting cells from damage, and managing specific symptoms. The disclosure therefore specifically suggests the following medications and therapeutic approaches when a patient with Crohn’s disease is likely to be suffering from mitochondrial dysfunction, manifested by a decrease in serum citrulline levels.Administration of Nutritional Supplements and Vitamins

[0053] Coenzyme Q10 (CoQ10) is a vital component of the electron transport chain and helps to improve mitochondrial function. Riboflavin (vitamin B2) is essential for mitochondrial energy production. Carnitine is required in the transport of fatty acids into mitochondria for energy production. Thiamine (vitamin B1 ) is involved in energy metabolism and mitochondrial function. Vitamin E is known to act as an antioxidant for protecting cells from oxidative damage. Alpha-lipoic acid is another antioxidant that can help protect mitochondria, and creatine can help improve muscle function, thus strengthening mitochondrial function. Levo-carnitine (L-carnitine) helps in the metabolism of fatty acids and can alleviate symptoms related to mitochondrial dysfunction.

[0054] The administration of the amino acids arginine and citrulline is well known for managing stroke-like episodes in MELAS syndrome (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes) whereas the administration of taurine can help in mitochondrial myopathies. Taurine occurs naturally in fish and meat, but the daily intake may be reduced by vegetarian or vegan diets. Additionally, taurine is partially destroyed by heat in processes such as baking and boiling. Either raw feeding or addition of taurine can satisfy this requirement. Prematurely born infants are believed to lack the enzymes needed to convert cystathionine to cysteine, and may, therefore, become deficient in taurine. Taurine is present in breast milk, and has been added to many infant formulas, as a measure of prudence since the early 1980s. Taurine is not regarded an essential human dietary nutrient as high-quality clinical studies on the effects of taurine in the body following dietary supplementation are absent. Notwithstanding, its use as a supplement is an option to treat mitochondrial dysfunction in patients with Crohn’s disease.

[0055] Another option of managing a mitochondrial dysfunction is a high-fat, low- carbohydrate diet (ketogenic diet) which provides an alternative energy source in the body. Another potentially useful dietary approach is the Modified Atkins diet. Ketogenic diets are used to treat epilepsy in children and in adults too, where it has a positive effect in reducing seizures. There is some evidence that therapeutic ketogenic diets, and that a less strict regimen, such as a modified Atkins diet, is effective in reducing flares in Crohn's disease and like regular aerobic exercise which can help improve mitochondrial function and overall health. In summary, these results therefore suggest determining first the respective vitamin statuses and possible nutritional deficiencies in patients with Crohn’s disease before any other therapy.Antioxidants and Pharmacological Agents

[0056] Mitochondrial dysfunction is known to result in the production of an abnormally high amount of reactive oxygen and nitrogen species, which results in redox imbalance and glutathione deficiency. Therefore, another option of treating mitochondrial dysfunction comprises the administration of antioxidants. A useful antioxidant is for example Idebenone®, which is a synthetic analog of CoQ10 that acts as an antioxidant and inhibitor of lipoperoxide formation. It is further a transporter in the electron transport chain of mitochondria. This action increases the production of adenosine triphosphate (ATP) which is the main energy source for cells. Another potent antioxidant is EPI-743 which has already been investigated for mitochondrial disease treatment. Therapeutics such as EPI-743 (a-tocotrienol quinone) and RP103 (cysteamine bitartrate) have the theoretical potential to improve redox imbalance by increasing intracellular glutathione (cf. Enns GM, Cohen BH. Clinical Trials in Mitochondrial Disease: An Update on EPI-743 and RP103. Journal of Inborn Errors of Metabolism and Screening. 2017;5. doi:10.1177 / 2326409817733013).

[0057] A potentially useful drug is dichloroacetate (DCA) because it can inhibit the enzyme pyruvate dehydrogenase kinase and reduce lactic acid levels in patients with mitochondrial disorders. Dichloroacetate (DCA) is well tolerated and effective in adults with lactic acidosis. Elamipretide (SS-31 ) is a small mitochondrially-targeted tetrapeptide (D-Arg- dimethylTyr-Lys-Phe-NH2) that appears to reduce the production of toxic reactive oxygen species and stabilize cardiolipin (1 ,3-bis(sn-3’-phosphatidyl)-sn-glycerol), a component of the inner mitochondrial membrane where it constitutes about 20% of the total lipid composition. Most importantly, diabetes can also be the etiology of mitochondrial disorders and therefore anti-diabetic medication also represents a pharmacological treatment of Crohn’s disease and its flares and convulsions.

[0058] These suggested treatments must of course be tailored to the individual needs of the patient, and the management plan may involve a combination of the above approaches. It is important for patients with mitochondrial diseases to work closely with a team of healthcare providers, including neurologists, geneticists, and metabolic specialists, to develop an effective treatment plan.SYNOPSIS

[0059] The present application therefore provides a colorimetric assay and early biomarker for patient monitoring and medical treatment of patients suffering from a possible mitochondrial dysfunction, inflammatory bowel disease, particularly Crohn’s disease. The colorimetric assay measures the level of L-citrulline in a plasma or serum sample; and when the level of L-citrulline in the plasma or serum decreases or falls even below 30 pmol L- citrulline, this indicates a mitochondrial cell disorder and a relapse or increase in intestinal inflammation due to a flare of Crohn’s disease. A method of detecting and treating the mitochondrial dysfunction is provided too.

Claims

CLAIMS1. A method for monitoring patients suffering from Inflammatory Bowel Disease, in particular Morbus Crohn, which is characterized by the steps: -(i) obtaining an in-vitro sample of plasma or serum from the patient;(ii) measuring the concentration of L-citrulline in said plasma or serum sample;(iii) comparing the concentration of L-citrulline found in the plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline indicates a mitochondrial cell disorder and a relapse or increase in Crohn’s disease.

2. The method of claim 1 , wherein a measured L-citrulline concentration below 30 pmol / L in the patient’s plasma or serum indicates mitochondrial dysfunction and inflammation.

3. The method of claim 1 or claim 2, wherein a decreased L-citrulline concentration below 30 pmol / L in the patient’s plasma or serum indicates a need to support mitochondrial function by providing the patient’s diet with an alternative energy source, additional antioxidants, nutritional supplements and vitamins.

4. The method of claim 1 or claim 2, wherein a measured L-citrulline concentration below 30 pmol in serum or plasma of a patient with Crohn’s disease indicates a relapse or increased inflammation due to this disease.

5. The method according to any preceding claims 1 to 4, wherein the L-citrulline concentration in the patient’s plasma or serum is determined using tandem mass spectrometry, amino acid profiling, and / or immunoassay.

6. The method according to any preceding claims 1 to 4, wherein the L-citrulline concentration in the patient’s plasma or serum is determined by a colour reaction.

7. The method according to claim 6, wherein the plasma or serum sample is subjected to an enzymatic treatment with urease prior to the colour reaction.

8. The method according to claim 6 or claim 7, further comprising a deproteination treatment before the colour reaction.

9. A method of monitoring patients suffering from Morbus Crohn or inflammatory bowel disease comprising the steps of: -(i) obtaining an in-vitro sample of plasma or serum taken from a patient;(ii) subjecting the sample to enzymatic treatment with urease to render it substantially urea free;(iii) subjecting the sample to a deproteination treatment to obtain a substantially protein-free liquid sample;(iv) sequentially adding (A) an aqueous solution containing diacetyl monoxime (DAMO) and thiosemicarbazide (TSC) and (B) an acidic solution containing a catalytically active iron or manganese redox agent; and(v) heating the sample mixture to above 90 degrees Celsius to form triazine chromophores having peak absorbance in the range of 530 to 540 nm; and(vi) measuring the colour intensity at 530 or 540 nm in comparison with a series of L-citrulline standards in a salt solution to determine the concentration of L-citrulline in the plasma or serum sample; and(vi) comparing the concentration of L-citrulline found in said plasma or serum sample with a previously measured concentration of L-citrulline in the patient’s plasma or serum, wherein any decreased concentration of L-citrulline indicates a mitochondrial disorder and relapse or an increase in Crohn’s disease.

10. The method of claim 9, wherein solution A contains diacetyl monoxime and thiosemicarbazide in a molar ratio of 30 to 70, preferably 40 to 50.11 . The method of claim 9 or claim 10, wherein solution B contains 3 M phosphoric acid, 6 M H2SO4, and 0.1 to 5 mM NH4Fe(SO4)2, preferably 2 mM NH4Fe(SO4)2.

12. The method of any claim 9 to 11 , solution A and solution B are mixed in a ratio of 1 : 2 , 5.

13. The method of any claim 9 to 12, wherein the volume of the plasma or serum sample to the volume of the urease solution is greater than 20:1 .

14. A kit of parts for photometric testing and monitoring of patients suffering from inflammatory bowel disease, comprising:- an aqueous urease concentrate for treating and removing any substantial urea from the plasma or serum sample without diluting the sample by more than 5 percent, a concentrate solution of trichloroacetic acid for deproteinization of the sample without diluting the sample by more than 10 percent, a solution (A), containing diacetyl monoxime and thiosemicarbazide, and an acidic solution (B) containing a catalytically active iron- or manganese-containing redox agent suitable for the synthesis of triazine chromophores of L-citrulline having a peak absorption in the range of 530 to 560 nm, and one or more aqueous standard solutions of L-citrulline, suitable for colorimetric determination of L-citrulline in plasma or serum in the concentration range from 0.5 to 100 pmol / L.

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