WO2009109647A1

WO2009109647A1 - Method for monitoring a metabolic state by measuring inositol phosphate

Info

Publication number: WO2009109647A1
Application number: PCT/EP2009/052645
Authority: WO
Inventors: Gunnar Norstedt; Petra Tollet Egnell; Thomas Moritz; Suad Efendic
Original assignee: Gunnar Norstedt; Petra Tollet Egnell; Thomas Moritz; Suad Efendic
Priority date: 2008-03-05
Filing date: 2009-03-05
Publication date: 2009-09-11

Abstract

A method of monitoring the metabolic state in a mammal, comprising obtaining a test sample from the mammal and measuring the amount of inositol phosphate in the sample. A kit for use in such a method.

Description

METHOD FOR MONITORING A METABOLIC STATE BY MEASURING INOSITOL PHOSPHATE

Field of the invention

The present invention generally relates to methods for monitoring the metabolic state of a mammal.

Background of the invention

Diabetes is a common condition that is caused by an insufficient production or sensitivity to insulin. Diabetes is treated in different ways; in severe cases insulin injections are needed whereas in milder cases advice on life style changes are sufficient. Such life style changes include diet changes as well as the important component of increased physical activity.

An important diagnostic test of diabetes is measurement of glucose levels in blood or urine. Measurement of glucose is an essential component not only to diagnose diabetes but also to follow effects of treatments.

During recent years one has realized that diabetes exists before an overt clinical picture is at hand. Such forms of pre-diabetes can e.g. be detected by following the duration of an oral glucose load but there are forms of pre-diabetes, e.g. hepatic insulin resistance that require even more specialised techniques to be detected. In the field of diabetes there is a need to find simple ways to diagnose pre-diabetes or risk for diabetes and there is also a need to improve methods to determine if treatment regimens really are effective.

Within the field of diabetology there is a consensus that it is very important to find ways for early diagnosis as well as to be able to find markers that reflect improvements in the diabetic state because early treatment significantly delays overt diabetes which in many cases develop into a severe and complicated disorder.

Measurements of glucose are used in routine clinical practice and this is an important component in the diagnosis and treatment of diabetes. It is however relevant to note that also other markers for diabetes exist. Some uncommon forms of diabetes can be detected using genetic analysis, however most common forms of diabetes cannot be genetically demonstrated because of environmental causes. In common forms of diabetes there are other known metabolic perturbations notably related to blood lipid profiles.

WO9960406A1 relates to methods of assessing glycemic control in mammals with Type II diabetes by measuring the level of myo-inositol in body fluid, e.g. urine or blood.

Summary of the invention

According to one aspect the present invention provides a method of monitoring the metabolic state in a mammal, comprising obtaining a test sample from the mammal and measuring the amount of inositol phosphate in the sample.

In one embodiment, the invention provides a method of predicting a condition related to insulin insensitivity in a mammal or of following treatment effects in a condition related to insulin insensitivity in the mammal comprising obtaining a test sample from the mammal and measuring the amount of inositol phosphate in the sample.

In one embodiment, the mammal is selected from a diabetic subject, a pre-diabetic subject, a subject exposed to metabolic risk factors, or a physically inactive subject.

In one embodiment, the invention provides a method of deteπniriing whether there is a change, e.g. an improvement, of the metabolic state of a subject receiving medical treatment, and/or treatment comprising dietary change and/or physical exercise.

According to another aspect, the present invention provides a kit for use in a method as defined herein above.

According to still another aspect, the present invention provides a system for monitoring the metabolic state in a mammal, comprising obtaining a test sample from the mammal, measuring the amount of inositol phosphate in the sample, comparing the measured value of the amount of inositol phosphate to at least one reference value, and deteπnining the metabolic state or the change in metabolic state of the mammal based on the comparison.

Further embodiments of the invention will be apparent from the following description. Brief description of the drawings

Figure 1 is a diagram showing correlation analysis between GC/MS-derived peak area for inositol- 1 -phosphate and insulin sensitivity (M value) determined in diabetic men participating in training study 1 (A) or training study 2 (B); Figure 2 is a chromatogram of myo-inositol; and

Figure 3 is a chromatogram of myo-inositol mono phosphate.

Detailed description of the invention

In the context of the present invention, and unless otherwise specified or apparent from the context, the "metabolic state" of an individual is to be understood as referring to the state of glucose regulation capacity of the individual. An impaired glucose regulation measured by e.g. Impaired Glucose Tolerance (IGT) and Impaired Fasting Glycaemia (BFG) is a metabolic read out that defines an intermediate state between normal glucose homeostasis and diabetes.

The effect of physical training on the state of diabetes has been studied. The patients were put in a physical training program that is outlined below. A number of parameters were measured before and after the study period. A principal conclusion was that physical training is a highly effective method to improve the state of diabetes.

In addition to conventional methods to determine the metabolic state the present inventors analysed serum for a large number (approx. 2000) metabolites. It was surprisingly found that one compound, inositol- 1 -phosphate, correlated well with an improved metabolic state following training.

In the present invention, the use of inositol phosphate measurements as a marker for an improved metabolic state after physical exercise is described. This marker can be used in isolation or together with other metabolic markers to predict or follow treatment effects in diabetes, in diabetes-related disorders or in untrained individuals to signal metabolic improvements. The existing metabolic markers that can be combined with inositol phosphate measurements include clinical chemistry measurements e.g. glucose, cholesterol, lipoproteins, insulin, insulin C peptide, IGF binding proteins, notably IGF-BPl, liver enzymes as well as clinical physiology recordings of heart/lung functions e.g. pulse rate, blood pressure, EKG, oxygen uptake and muscle strength. In addition to using inositol phosphate measurements in combination with other markers that signal metabolic disorders it is well known to persons skilled in the art that measurements need to be put in reference to either the volume or to a reference compound that preferably is not altered by the disease state. In the present invention such reference compounds include any compound not altered by diabetes as well as reference compounds altered by diabetes put as a ratio to inositol phosphate.

The existence of inositol phosphates inside cells is well known and in fact these molecules have an important role in intra cellular signalling (review [I]), however the present disclosure that inositol phosphate is useful as a marker of the metabolic state of a mammal is a surprising finding. To the best knowledge of the present inventors, inositol phosphate has not hitherto been suggested as such a marker, although one patent application (United States Patent

5183764 [2]) describes the use of inositol measurements in the context of diabetes. Inositol phosphate is however a different type of molecule compared to inositol.

There is a distinct chemical difference between inositol and inositol phosphate. In addition, these two compounds behave completely different in the context of serving as markers for insulin resistance. In the US application by Kennington et al (US 5,183,764) it is described that low levels of inositol in urine or serum indicate insulin resistance. In the present application low levels of inositol phosphate, on the contrary, indicate improvement of insulin resistance.

The phosphate group of inositol phosphate in nature is located at different positions on the molecule; the present invention preferentially concerns inositol- 1 -phosphate but also other phosphate groups combined or not combined with inositol- 1 -phosphate. For the purpose of the present invention, therefore, the term "inositol phosphate" refers to any inositol carrying a phosphate group attached at any carbon of the inositol molecule, but preferably to carbon I₅ i.e. inositol- 1 -phosphate.

For the purpose of the present invention, the term "marker" is defined as a measurable substance that can serve as a diagnostic read out of medical use. In its most commonly used sense, the marker will deliver a signal, measurable in a body fluid, of relevance for a pathological state. Although inositol phosphate can be measured in cell or tissue extracts such use does not fall within the scope of the present patent application. The tissue sources for inositol phosphates are at the present not known. In analogy the molecular mechanism(s) that explains the utility of inositol phosphates as suitable markers for diabetes are not known, nor is it known whether inositol phosphate exists in free forms in the mammal body, or is bound to e.g. proteins. This lack of knowledge does however not impinge on the utility of inositol phosphate measurements in its function to serve as a marker of metabolic disturbances.

In the below presented examples the utility of inositol phosphate measurements in blood samples are shown. To persons skilled in the art it is well known that blood can be fractionated into e.g. plasma or serum. In addition, other body fluids are routinely used in the field of diagnostic markers; these include e.g. urine, saliva, intraperitoneal fluid, and methods and systems using any of these body fluids fall within the scope of the present invention.

In one embodiment of the present invention a blood sample from a patient (diabetic, pre- diabetic or patient at risk of developing diabetes) is collected after which inositol phosphate is measured. By comparing to normal levels, statements can be made of metabolic derangements. As known by persons of skill within the medical field, e.g. physicians, measurements of single metabolites can have added value if such readings are related to other components routinely measured in patient blood.

In another embodiment, therefore, measurement of inositol phosphate in blood samples is made before and during treatment to follow the efficacy of therapy. Such treatment includes both changes in life style (diet/exercise) and conventional drug therapy.

In still another embodiment, inositol phosphate measurements are used in order to group or classify diabetic subjects to guide further therapy or to test drug safety or drug efficacy studies; the latter refers to the situation that improvements of insulin sensitivity is a common goal for drug development and that unwanted side effects of certain drugs can include the generation of insulin resistance.

The term metabolic disorders includes several disorders e.g. diabetes type I and type II, adiposity and age related disorders, lipid related disorders, disorders related to endocrine disturbances e.g. reduced levels of growth hormone, thyroid hormone and/or androgen or polycystic ovary disease. According to one aspect the present invention provides a method and system for monitoring the metabolic state in a mammal, comprising obtaining a test sample from the mammal and measuring the amount of inositol phosphate in the sample.

This embodiment may comprise obtaining a sample of a bodily fluid from a mammal; measuring the amount of inositol phosphate in the sample; comparing the amount of inositol phosphate in the sample to a defined reference level of inositol phosphate indicative of a defined metabolic state; and assessing the change of metabolic state in the mammal based on this comparison.

In this embodiment, also the sample content of one or more other metabolic markers may be measured, e.g. glucose, cholesterol, lipoproteins, insulin, insulin C peptide, IGF binding proteins, notably IGF-BPl. Additionally, liver enzymes as well as clinical physiology recordings of heart/lung functions e.g. pulse rate, blood pressure, EKG, oxygen uptake and muscle strength may be effected.

Additionally, the content of a reference compound in the sample may be determined, the contents of which reference compound in the body of the mammal is not related to the metabolic state of the mammal. Many such endogenous compounds can be found in body fluids both within the category of compounds referred to as proteins or within the category referred to as low molecular weight metabolites.

The defined metabolic state may be e.g. a diabetic, pre-diabetic state, such as a state of impaired insulin sensitivity; but also may be a normal metabolic state.

The reference level of inositol phosphate may be a concentration of inositol phosphate in a sample obtained from the subject at the beginning of e.g. a therapeutic treatment regimen, e.g. a regimen of physical exercise and/or drug treatment.

The change of metabolic state may be e.g. an improved insulin sensitivity or an impaired insulin sensitivity. The system and method of the present invention advantageously permits to predict a condition related to insulin insensitivity in a mammal or to follow the effects of therapeutic treatment of a condition related to insulin insensitivity in the mammal.

Thus, by the method and system of the invention, the physician and the subject being followed will be provided with a sensitive and reliable indication of e.g. the effect of an ongoing treatment or the necessity for undertaking treatment or for other measures, such as change of lifestyle, diet or necessity of medication, or for change of medication.

In one embodiment, the mammal is selected from a diabetic subject, a pre-diabetic subject, a subject exposed to metabolic risk factors, or a physically inactive subject. The mammal may be an animal, e.g. a domestic, farm or laboratory animal, such as a dog, cat, horse, cow, sheep, rabbit, rat or mouse, or a human.

According to one embodiment, a method as defined herein above comprises:

- obtaining a bodily fluid sample, e.g. a blood sample, from the mammal subject, the metabolic state of which is to be determined;

- determining the amount of inositol phosphate in the sample; and

- relating the amount of inositol phosphate in the sample to a reference parameter, whereby a change in the amount of inositol phosphate compared to the reference parameter is indicative of a change in the metabolic state of the mammal subject; e.g. a decrease in the amount of inositol phosphate compared to a reference parameter is indicative of an improved metabolic state, and vice versa.

The reference parameter may be e.g. an amount of inositol phosphate determined in a sample from a mammal subject that is to undergo a treatment, before initiating the treatment, or may be e.g. the amount, in the same or another sample, of another component of the bodily fluid of the mammal subject.

For example, a method as defined herein above may comprise:

- contacting the sample with an antibody specific to inositol phosphate under conditions suitable for the antibody to bind to the inositol phosphate in the sample; - detecting the level of antibody bound to the inositol phosphate, wherein the level of the inositol phosphate in the sample is determined by detecting the level of antibody bound to the inositol phosphate; and

- comparing the level of the inositol phosphate in the sample to a defined reference level of the inositol phosphate, wherein a reduced level of the inositol phosphate is indicative of an improved metabolic state.

According to another aspect, the present invention provides a diagnostic kit for use in a method and system as defined herein above.

For example, a diagnostic kit permitting to determine the presence at least one sample from the mammal subject may be of the flow test strip type as well-known and commonly used. Such test strips allows, simultaneous, specific qualitative or semi-quantitative detection of one or several analytes on the same strip, using e.g. urine, saliva, serum, plasma, or whole blood as sample.

Further embodiments of the invention will be apparent from the following description.

Example 1 The selection of inositol phosphate as a marker from a large number of other metabolites

Patients: 12 diabetic patients and 11 healthy subjects took part in a group training program, three times a week for 12 weeks. Various clinical tests were performed before and after training including aerobic capacity, body composition and the measurement of different metabolic parameters including insulin sensitivity. Training improved whole body insulin sensitivity determined by hyperinsulinemic euglycemic clamps. In men the glucose infusion rates (M values) required to maintain euglycemica were increase approximately 100% in diabetic men after training, approaching the values obtained for healthy subjects. In summary the results show that 12 weeks of moderate taining improves insulin sensitivity in diabetic men in spite of unaltered aerobic capacity or body composition.

Metabolomics: Fresh EDTA anticoagulated blood was collected and plasma was prepared by centrifuging at 160Og for 10 min at 4⁰C, and stored at -8O⁰C. Low molecular weight compounds (LMC) from the plasma samples (100 μl) were extracted and de-proteinization with a 900-μl mixture of methanol and water (8:1 v/v). A 200 μl aliquot of the supernatant was then transferred to a GOMS vial and evaporated to dryness. Prior to GC/MS analysis, the samples were methoxymated at room temperature for 16 h (with 30 μl of 15 mg/ml methoxyamine in pyridine) and trimethylsilylated with 30 μl of MSTFA with 1% TMCS as catalyst for 1 h. After silylation, 30 μl of heptane containing 45 ng/μl methyl octadecanoate was added.

The samples were analyzed by GC/TOFMS together with blank control samples and n- alkanes (C₁₂-C₄₀) series for calculation of retention index (Schauer et al., 2005 [3]). One μl of each derivatized sample was injected splitless by an Agilent 7683 autosampler (Agilent,

Atlanta, GA, USA) into an Agilent 6890 gas chromatograph equipped with a lθ m x θ.18 mm i.d. fused silica capillary column with a chemically bonded 0.18 μm DB 5-MS stationary phase (J&W Scientific, Folsom, CA, USA). The injector temperature was 270⁰C, the septum purge flow rate was 20 ml min^"1 and the purge was turned on after 60 s. The gas flow rate through the column was 1 ml min^"1, the column temperature was held at 70⁰C for 2 minutes, then increased by 40⁰C min^"1 to 320⁰C, and held there for 2 min. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer, GC/TOFMS (Leco Corp., St Joseph, MI, USA). The transfer line and the ion source temperatures were 250⁰C and 200⁰C, respectively. Ions were generated by a 70 eV electron beam at an ionization current of 2.0 mA, and 30 spectra s^"1 were recorded in the mass range 50 to 800 m/z. The acceleration voltage was turned on after a solvent delay of 170 s. The detector voltage was 1720 V.

Results: The mass spectrometry results, shown in Fig. 4, indicate that specific metabolites correlated to the improvement of insulin sensitivity. In fact 18 different differences were found to be related to improved metabolic state. Untrained subjects and insulin insensitivity could be related to glucose, cholesterol, inositol- 1 -phosphate and pyroglutamic acid and were thus reduced in the circulation of diabetic men after training. Other molecules were increased including stearic acid, 3β-hydroxybutyric acid and succinic acid. Inositol- 1-phophate showed the highest correlation to insulin sensitivity in diabetic men whereas an as yet unknown fatty acid correlated best with insulin sensitivity in women. The observation that inositol- 1- phsophate negatively correlated with insulin sensitivity in diabetic men was validated using samples obtained from another training study on 15 diabetic men (Fig 1). As previously stated the combination of inositol phosphate with metabolites that increase and or decrease can strengthen the diagnostic value of inositol phosphate measurements and this also include the use of inositol as a metabolite to be put in relation to inositol phosphate. For further details of this example the reader is referred to the publication: J KuM, T Moritz, H Stenlund, K Lundgren, P Bavenholm, S Efendic, G Norstedt and P Tollet-Egnell Metabolomics as a tool to evaluate exercise-induced improvements in insulin sensitivity Metabolomics, 2008, 4:273- 282 included herein as supporting material (6).

Example 2 LC-ESI/MS/MS assay to detect and quantify inositol phosphate

In this example inositol phosphate is measured using similar principles (mass spectrometry) as described above but an assay is developed to detect inositol and inositol phosphates in an unconjugated manner.

Fresh EDTA anticoagulated blood was collected and plasma was prepared by centrifuging at 160Og for 10 min at 4⁰C, and stored at -8O⁰C. Proteins from 50-100 μl plasma were precipitated using 200 μl acetonitrile, vortex mixed and centrifuged (10 min at 16K x g). The supernatant containing 150 μl of the supernatant was transferred to capped glass autosampler vials to be injected into the LC-MS system. Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C8 (4.6 x 100 mm, 5 urn) column manufactured by Agilent. Two mobile phase conditions were developed, as an isocratic elution containing 80% acetonitrile (ACN) with 0.1% formic acid in water for wryσ-inositol and a gradient elution for myoinositol mono phosphate measurement. For gradient elution, mobile phases were (A) 0.1% formic acid in water and (B) acetonitrile. Gradient conditions were as follows: 5% B for 1 min, increased to 95% B over 3 min and re-equilibrated at 5% B for 4 min. The column oven temperature, injection volume and flow rate were set at 40 C, 5 μL and 450 μl/min, respectively. The LC system was connected to the mass spectrometer via electrospray ionization (ESI) interface and was controlled by the Analyst 1.4.1 software designed by Applied Biosystem, Canada.

The mass spectrometry analysis was performed on a SCEEX 2000 tandem mass spectrometer (Applied Biosystem, MDS Sciex, Ontario, Canada). Ionization was achieved in the negative ion mode by the ESI interface, and data acquisition done in the selected reaction monitoring (SRM) mode. The optimal conditions for wryø-inositol were: the mass spectrometric transition m/z 179 (MS), ion spray voltage -4500V, nebulizing gas (gas 1) and auxiliary gas (gas 2) 35 and 45 psi, source temperature 500 ⁰C, and curtain gas 35 psi. The declustering potential, focusing potential and entrance potential were at -35 V, -300 V and -10 V₅ respectively. The dwell time and inter channel delay were set to 500 and 5 msec to monitor the precursor ion at m/z 179 for /wyo-inositol. The optimal conditions for myoinositol mono phosphate were: mass spectrometric transition at m/z 259/79 (MS/MS), ionspray voltage -4500V, nebulizing gas (gas 1) and auxiliary gas (gas 2) 45 psi, source temperature 500 ⁰C, curtain gas 35 psi, and CAD gas 10 psi. The declustering potential, focusing potential and entrance potential were at -26 V, -310 V and -10 V, respectively. The collision energy was maintained at -25, dwell tune and inter channel delay were set to 500 and 10 msec to monitor the product ions at m/z 259/79 for /wyo-inositol salt. Two representative chromatograms extracted from blank serum sample are shown in Figs. 2 and 3.

The above described method can be used for quantification by running parallel standard curves with know amounts of inositol phosphate. Alternatively the ratio between inositol and inositol phosphate can be accurately determined. An accurate quantification of inositol phosphate can be achieved by using isotope labelled inositol phosphates.

Example 3 Outline of an immune assay for inositol phosphate measurements

This assay is based on antibodies, preferably monoclonal antibodies that specifically bind inositol phosphate. The generation of such antibodies is well known in the art and one example of a monoclonal antibody that reacts with inositol- 1 -phosphate has been described in a patent from Japan [4]. Alternatives to antibodies exist since the only function of such is to bind inositol phosphate and such reagents can be based on the affinity of inositol phosphate to metal or by the development of recombinant proteins engineered to bind inositol phosphate. Such techniques include phage display but also the use of protein A or ubiquitin where a portion of the protein is used to randomly alter the amino acid sequence, hi the context of the present application the preferred use is antibodies but also other proteins or compounds can be used as long as they bind inositol with a high affinity and a high specificity.

hi one embodiment of the present application, immune assays are carried out on patient plasma/serum/urine, either extracted as described in Example 2 or alternatively these assays can be used without extraction of the sample. A suitable sample volume is 100 μl plasma but the sample input can vary e.g. between 10-1000 μl. The sample to be tested for content of inositol phosphate is subsequently contacted with antibodies that specifically react with inositol phosphate, the concentration of such antibodies vary greatly dependent on titer but can be in the range of 1 : 1000 dilution of a polyclonal antiserum or in lower dilutions if monoclonal antibodies are used. Agents other than antibodies that bind inositol phosphate can be used as long as they bind with a high affinity and specificity. Such molecules can be recombinant proteins engineered to bind inositol phosphate or DNA/RNA aptamers engineered for the same purpose. The concentration of the binding entity should in general be in excess of the reactant (i.e. inositol phosphate) to be measured.

The test sample is then incubated for a predetermined length of time and temperature. The time and temperature depend on the source of test sample and on the antibody, normally an over night incubation at 4⁰C is used. The last step is to determine how much inositol phosphate that has been bound to the antibody. In one embodiment when the antibody is bound to a solid surface, the incubation is washed and the ability of the sample to compete with radiolabelled inositol phosphate is determined. As known to persons skilled in the art antibody based assays can be developed in several different ways, either by immobilizing the antibody or the reactant to the solid phase, by using radiolabelled ligands or secondary detections of the antibody and in some cases so called homogenous assays can be developed where the separations steps can be exchanged for measurements of proximity of radioactivity or fluorescence. Within the context of the present invention, these examples seeks to demonstrate the utility of a protein that binds inositol phosphate to develop an assay where multiple options exist e.g. ELISA, scintillation proximity assay, fluorescence quenching and so forth.

Example 4

Outline of assays that simplify inositol-l-phosphate recordings.

Simplicity and speed is a very important aspect of clinical measurements. A good example is blood glucose measurements were patients themselves can determine the level of blood glucose. The technological principle of the glucose oxidation method can be used for measurements of inositol phosphates if enzymatic principles only act on inositol phosphate. Biosensors based on antibody bindings e.g. Biacore (plasmon resonance) can be used as can other similar technologies. Alternative methods can depend on ability to detect photons or altered chemical properties in chemical reactions involving inositol phosphate. Since the synthesis and degradation of inositol phosphate is known such enzymatic assays can be developed.

In one embodiment, an assay as described in US patent application No. 10/539,544 [5] is used.

Example 5

Interpretation of inositol measurements alone or in combination with other measured parameters. As described above several techniques can be used to measure inositol phosphate and similar technologies can be developed once the nature of the, at the present unknown, fatty acid disclosed herein has been determined. In one embodiment of the present invention a subject will deliver blood samples where the content of inositol-phosphate will be determined using any of the above techniques. Preferably the identical sample is also used to analyse other well established metabolic markers (e.g. glucose, insulin, C- peptide, free fatty acids, and cholesterol). In one embodiment of the invention the levels of inositol- 1-phophate either alone or in relation to any of the metabolic markers exemplified is used as a marker of metabolic state; a high level of inositol- 1 -phosphate will indicate an increased risk for insulin insensitivity. As is known to persons skilled in the art, the quantity of inositol- 1 -phosphate that is to be regarded as normal or pathological needs to be determined in a very large material to capture the normal variation. At this stage it can be stated that levels of inositol phosphate in blood varies between sub ng/ml to μg/ml. In one embodiment records of inositol-phosphate will be taken before and after training in pre-diabetic, diabetic or normal subjects and the comparison of inositol phosphate before and after will indicate an effect of training; a reduction of inositol phosphate is expected if an improved metabolic state is achieved.

In Table 1, annotated or classified GC/TOFMS peaks explaining the difference in blood plasma between trained and non-trained T2D subjects . Table 1

GC/TOFMS peaks are listed which according to OPLS-DA were identified as important for explaining the difference in blood plasma between trained and non-trained diabetic subjects. Metabolites were analyzed as methyloxime-trimethylsilyl derivatives. ^aPeaks are named according to UPSC in-house mass spectra library. Annotation of peaks was performed by comparing mass spectrum and retention index (RIf with the UPSC in-house mass spectra library or the mass spectra library maintained by the Max Planck Institute (MPI) in GoIm (http://csbdb .mpimp-gokn.mpg.de/csbdb/gmd/gmd.html). ^dFirst loading vector (w[l]) from the OPLS-DA model between trained and non-trained subjects describes the importance of different GC/MS peaks for explaining the differences between the samples. Positive values are peaks correlated with trained T2D subjects (after exercise), negative values are peaks correlated with non-trained T2D subjects (before exercise). ^ePeaks annotated or classified according "MOOO..." are identical or similar to non-annotated mass spectra in the MPI- library. Naming refers to MPI-spectra numbering. UPSC mass spectra will shortly be available for download on UPSC homepage.

Example 6

Use of inositol-1-phosphate measurements to monitor alterations in cholesterol or lipoprotein metabolism

Metabolic disorders are frequently associated with alterations in cholesterol and lipoprotein metabolism. Different treatments are in current use to correct changes in lipid related disorders ranging from drugs to physical exercise. It is known that physical training exerts beneficial effects on body fat, blood pressure and dyslipoproteinaemia, which are common problems in subjects with reduced insulin sensitivity (such as patients suffering from type 2 diabetes, the metabolic syndrome or the poly cystic ovary syndrome). By testing patients before and after physical training we can demonstrate that training improves (reduces) VLDL- cholesterol in men with type 2 diabetes, and that this correlates strongly with reduced levels of inositol-1-phosphate. In one embodiment of the invention the levels of inositol- 1-phophate either alone or in relation to any of the metabolic markers exemplified is used as a marker of metabolic state; a high level of inositol-1-phosphate will indicate an increased risk for hypercholesterolemia and/or dyslipoproteinaemia.

As described herein, the present invention provides a sensitive and efficient system and method for monitoring the metabolic state in a mammal, as well as a kit for use in such a method. The invention will be of use to physicians and other persons in the health care sector, as well as to individual patients, who will be able to perforin inositol phosphate determinations themselves, e.g. by use of a kit according to the invention (cf glucose measurements). In the clinical situation, individuals with a proven or suspected metabolic disorder will be tested according to the present invention and this will assist in the diagnosis or to follow up therapy. Blood samples will be obtained preferably by using capillary blood or alternatively by collecting intravenous blood samples. Such samples, either to be used directly or to be used for preparation of plasma or serum will be used to measure inositol phosphate. The preferred measurement of inositol phosphate will be by use of a kit that is easy to use.

Alternatively, the samples will be sent to a clinical chemistry department, as available in most hospitals. The test results will be delivered to the health care person who can use the results by comparison with reference values. Alternatively, the multiple samples obtained before and during therapeutic interventions can be used to follow how effective the treatment is.

References

1. Shears, S. B., L. Yang, and X. Qian, Cell signaling by a physiologically reversible inositol phosphate kinase/phosphatase. Adv Enzyme Regul, 2004. 44: p. 265-77.

2. Kennington AS and Lamer J. Quatitative analysis for diabetic condition predictor involving chiro-inositol. United States Patent. No 5,183,764 dated Feb 2, 1998 3. Schauer N, Steinhauser D, Strelkov S, Schomburg D, Allison G, Moritz T, Lundgren K,

Roessner-Tunali U, Forbes MG, Willmitzer L, Fernie AR, Kopka J.

GC-MS libraries for the rapid identification of metabolites in complex biological samples.

FEBS Lett. 2005 Feb 28;579(6): 1332-7.

4. Monoclonal antibodies to inositol phosphate. Abe, Hayao; Takenawa, Tatatomi; Nakanishi, Osamu; Awaya, Akira. (Mitsui Toatsu Chemicals, Inc., Japan; Mitsui

Pharmaceuticals, Inc.). Jpn. Kokai Tokkyo Koho (1989), 11 pp. CODEN: JKXXAF JP

01168299 A 19890703 Heisei. Patent written in Japanese. Application: JP 87-327329

19871225. Priority: . CAN 112:176924 AN 1990:176924 CAPLUS Patent Family

Information Patent No. JP 01168299, A Dated 19890703 5. Bostwick; Robert ; et al. Inositol phosphate detection assays. US patent application No.

10/539,544 (Pub. No. 20060115863 Al) 6. J Kuhl, T Moritz, H Stenlund, K Lundgren, P Bavenholm, S Efendic, G Norstedt and P Tollet-Egnell Metabolomics as a tool to evaluate exercise-induced improvements in insulin sensitivity Metabolomics, 2008, 4:273-282

Claims

1. A method of monitoring the metabolic state in a mammal, comprising obtaining a test sample from the mammal and determining the amount of inositol phosphate in the sample.

2. The method according to claim 1, wherein the test sample is blood, serum, plasma, urine or saliva.

3. The method according to claim 1 or claim 2, wherein the sample volume is in the range of 1-1000 μl.

4. The method according to any of the claims 1-3, comprising additionally measuring at least one other metabolic marker of the mammal.

5. The method according to claim 4, wherein the at least one other metabolic marker is selected from glucose, cholesterol, lipoproteins, insulin, insulin C peptide, IGF binding proteins, e.g. IGF-BPl, liver enzymes, as well as clinical physiology recordings of heart/lung functions e.g. pulse rate, blood pressure, EKG, oxygen uptake and muscle strength.

6. The method according to any of the claims 1-5, performed to predict a condition related to insulin insensitivity or to follow treatment effects in a condition related to insulin insensitivity.

7. The method according to any of the claims 1-6, wherein the mammal is selected from a diabetic subject, a pre-diabetic subject, a subject exposed to metabolic risk factors, or a physically inactive subject.

8. The method according to claim 7, performed to determine any change of the metabolic state of a subject receiving medical treatment, and/or treatment comprising dietary change and/or physical exercise.

9. The method according to any of the claims 1-8, wherein the amount of inositol phosphate is determined by use of mass spectrometry.

10. The method according to any of the claims 1-8, wherein the amount of inositol phosphate is determined by use of an antibody that specifically binds inositol phosphate.

11. The method according to claim 10, wherein the antibody is monoclonal.

12. An antibody that specifically bind inositol phosphate for use in a method according to claim 10 or 11.

13. A diagnostic kit for use in a method according to claim 10, comprising an antibody according to claim 12.

14. A diagnostic kit additionally comprising means for determining the amount of glucose, cholesterol, lipoproteins, insulin, insulin C peptide, IGF binding proteins, e.g. IGF-BPl or liver enzymes in the sample.