Patent Application
20180372716
The present invention relates to methods of diagnosing disorders of the central nervous system (CNS) using metabolite profiling. In particular, the present invention relates to methods of diagnosing Multiple Sclerosis (MS) in human subjects.
Multiple sclerosis (MS) is the commonest cause of progressive disability in the Western world. From onset of the condition, the clinical phenotype of MS follows either a relapsing remitting (RR MS) course, or a progressive course (primary progressive (PP) MS). After a period of time in the RR phase the majority of patients develop a secondary progressive (SP) phase. It is during the progressive phases that the majority of disability accrues. It appears that inflammation predominates during the relapsing phase, whereas neurodegeneration is the pathological substrate of disability during the progressive phase. However, there is much overlap.
Unfortunately, current disease modifying agents targeting inflammatory mechanisms are not effective in progressive disease, and new treatment strategies are required. Exploring differences between the MS phenotypes may identify key mechanisms driving progression, and is the first step in developing effective new therapies to prevent disability.
The currently used main outcome measures in MS consist of clinical scales and magnetic resonance imaging (MRI). These are associated with a number of disadvantages. The clinical outcomes have low sensitivity and poor pathogenic specificity, and are open to subjective distortion due to patients and clinician subjectivity. A neuroprotective trial measuring clinical disability outcome may require 600 or more patients over two or even three years. MRI has been used to study inflammatory lesions and in studies of anti-inflammatory therapies. Atrophy is a surrogate marker for neuroprotection; however, it lacks sensitivity and is difficult to measure in patients with severe or advanced disease when the atrophy is marked. Additionally, pseudoatrophy is seen when inflammation is reduced such as with immunomodulatory drugs. Changes in myelin and damage in normal-appearing brain tissue are recognised pathologically, but are not easy to measure due to difficulty in measuring whole brain and spinal cord load.
Identifying specific biomarkers in biological fluids has been considered across a number of diseases, both in animal models and humans. Urines from patients with neurological disease, including MS, have been assayed for disease-specific markers, such as myelin basic protein-derived material indicating demyelination (Whitaker et al., 1994; Whitaker et al., 1995). However, these studies focused on the detection of a single specific metabolite, and although differences have been identified at a group level, predictive value at an individual level has been low.
There is therefore a need for an improved method of diagnosing MS that has an increased predictive value at an individual level. There is also a need to identify surrogate outcome measures of inflammation and neurodegeneration that may be used to power clinical studies of MS treatments. It would further be advantageous if the improved method could differentiate between different phases (also referred to as stages) of MS.
The present invention addresses one or more of the above problems by providing methods for diagnosing MS in humans.
In one aspect, the invention provides an in vitro method for diagnosing MS in a human test subject, comprising
Using a process of high resolution 1H-NMR (Nuclear Magnetic Resonance) spectroscopy, coupled with partial least squares discriminate analysis (PLS-DA), the present inventors have identified metabolites that can be used to diagnose MS, and further to differentiate between different specific disease phases of MS. The metabolites are small molecules that are intermediates and/or products of mammalian metabolism.
In one embodiment, said two or more (for example, two, three, or four) metabolites are selected from: blood metabolites, wherein said blood metabolites comprise:
phosphocholine, lactate, N-acetyl species, and beta-hydroxybutyrate; and/or urine metabolites, wherein said urine metabolites comprise: TMAO, citrate, creatinine, and lactate.
In another embodiment, said two or more metabolites (for example, two, three, four, five, or six) are selected from:
blood metabolites, wherein said blood metabolites comprise: phosphocholine, lactate, N-acetyl species, beta-hydroxybutyrate, fatty acid (preferably fatty acid δx-y0.88, 1.30, 5.35), and glucose; and/or urine metabolites, wherein said urine metabolites comprise:
TMAO, citrate, creatinine, and lactate.
In one embodiment, said two or more metabolites are selected from: blood metabolites, wherein said blood metabolites comprise:
L-glutamine, alanine, glucose, phosphocholine, fatty acid, N-acetyl species, lactate, and beta-hydroxybutyrate; and/or urine metabolites,
wherein said urine metabolites comprise:
TMAO, citrate, creatinine, and lactate.
In one embodiment, “N-acetyl species” means at least one of the following metabolites: N-acetyl aspartate, and N-acetyl glycoprotein.
In one embodiment, “glucose” comprises alpha-glucose and beta-glucose.
In one embodiment, glucose is alpha-glucose.
In one embodiment, glucose is beta-glucose.
In one embodiment, said blood metabolites further comprise a singlet (δx-y3.37) and a singlet (δx-y2.65) as measured using 1H-NMR.
In one embodiment, said metabolites are associated with the following parameters, as measured using 1H-NMR (Key: δ—chemical shift, M—multiplicity, A—Assignment of proton, s—singlet, d—doublet, t—triplet, q—quartet, dd—double doublet, ddd—double double doublet, m—multiplet, br—broad resonance, δx-y centre of the bin identified by SIMCA. Resonance values (chemical shift) are quoted relative to the methyl resonance of 3-trimethylsilyl-1-propionate (TSP) defined at 0.00 ppm, and are quoted with an error margin of ±0.01 ppm):
alanine (δ1.46, d, CH3; δ3.73, q, CH), fatty acid (δ0.90, br, CH3, δ1.30, br, CH2CH2CH3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), α-glucose (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H), β-glucose (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ1.63, d, C(1)H), lactate (δ1.32, d, CH3, δ4.11, q, CH), N-acetyl species (δ2.03, br s, COCH3), β-hydroxybutyrate (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), phosphocholine (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), L-glutamine (δ0.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), unidentified singlet (δx-y3.37), unidentified singlet (δx-y2.65), citrate (δ2.50, d, CHAHB, δ52.65, d, CHAHB), creatinine (δ53.05, s, NCH3, δ4.10, s, CH2), lactate (δ1.32, d, CH3, δ4.11, q, CH), and trimethylamine N-oxide (TMAO) (δ3 26, s, ON(CH3)3).
The present inventors have discovered that differences in the concentrations of the above metabolites, as compared to a specific reference standard, are present in blood and/or urine of subjects having MS (and in particular, MS of a specific disease phase), and that determining the concentrations of said metabolites in blood/urine from a test subject can be used to diagnose MS. Thus, the method of the present invention can be used to diagnose MS and to differentiate between disease phases of MS. In one embodiment, the method of the invention permits diagnosis of a specific disease phase of MS.
As used herein in relation to MS, “disease phase” refers to the specific MS clinical phenotypes of relapsing remitting (RR) phase MS, primary progressive (PP) phase MS, and secondary progressive (SP) phase MS. Said clinical phenotypes describe the disease course from onset.
The sample that is to be tested using the method of the invention can be derived from blood or urine that has been obtained from a human test subject.
The term blood comprises whole blood, blood serum (henceforth “serum”) and blood plasma (henceforth “plasma”). Serum and plasma are derived from blood and thus may be considered as specific subtypes within the broader genus “blood”. Processes for obtaining serum or plasma from blood are known in the art. For example, it is known in the art that blood can be subjected to centrifugation in order to separate red blood cells, white blood cells, and plasma. Serum is defined as plasma that lacks clotting factors. Serum can be obtained by centrifugation of blood in which the clotting process has been triggered. Optionally, this can be carried out in specialised centrifuge tubes designed for this purpose.
A sample for use in the method of the present invention can be derived from blood or urine that has undergone processing after being obtained from a test subject.
Alternatively, a sample can be derived from blood or urine that has not undergone any processing after being obtained from a test subject.
The method of the present invention thus encompasses the use of samples that have undergone minimal or zero processing before testing. This provides a significant advantage over prior art methods in terms of time, cost and practicality. By way of example, a urine sample obtained from a test subject may be tested directly using the method of the present invention, without further processing. Serum and plasma samples can be readily obtained from blood samples using simple and readily available techniques that are well known in the art, as described above.
The method of the present invention is an in vitro diagnostic method. Thus, the method of the present invention can be carried out in vitro on an isolated sample that has been obtained from a test subject.
The method of the invention provides a number of advantages. The use of samples derived from tissue fluids that are readily accessible allows for samples to be measured longitudinally. Furthermore, the method of the invention can advantageously be used to study of groups of patients with pure neuroprotective conditions, pure inflammatory conditions and different phases and types of MS, thus permitting the separation out of pathological processes for measurement. For example, a useful outcome would be “a move from the relapsing remitting phase of MS to the secondary progressive phase” (the former early stage causes little permanent disability whereas the latter is where the disability is progressively accrued). At present this transfer is gradual and not clinically clear, such that a date can only be retrospectively identified and only within 12-18 months. Thus, by analysing the biomarkers of these two phases using the method of the invention, it is possible to use a time to transfer as an outcome. Additionally, by using the method of the invention to separate out early versus late MS, it is possible to identify differentiating markers that can measure disease progression. Similarly, the method of the invention can be used to identify diagnostic specific features relevant to MS versus other inflammatory conditions and the same with neurodegenerative conditions. This provides the identification of relevant aetiological markers that can be used diagnostically, and of treatment targets. Thus, the method of the invention also advantageously provides new biomarkers for evaluating neuroprotective treatments.
Step (i) of the method as described above comprises (or consists of) determining the concentrations of two or more metabolites in the diagnostic sample, wherein said two or more metabolites are selected from blood metabolites and urine metabolites as described above. A blood metabolite is a metabolite that can be identified in blood (including serum and plasma). A urine metabolite is a metabolite that can be identified in urine. The metabolite lactate has been identified by the present inventors in both serum and urine and is thus classed as both a blood metabolite and a urine metabolite.
The present inventors have discovered that results having high utility (such as high accuracy and high predictive value) require the concentrations of at least two metabolites to be determined. Prior art methods that measure only a single metabolite have been found to lack accuracy and predictive value.
The concentrations of the metabolites in the sample (as described above) can be determined using any suitable technique known in the art. By way of example, the following techniques can be used to detect and quantify small molecules in solution, and are thus suitable for determining metabolite concentrations: Nuclear Magnetic Resonance (NMR) spectroscopy, mass spectrometry, gas chromatography, ultraviolet (UV) spectrometry (for example in combination with high-performance liquid chromatography [HPLC] as HPLC-UV), and infrared spectroscopy.
In one embodiment, the concentrations of said two or more metabolites are determined using NMR spectroscopy. In one embodiment, the concentrations of said two or more metabolites are determined using mass spectrometry. In one embodiment, the concentrations of said two or more metabolites are determined using HPLC-UV. In one embodiment, the concentrations of said two or more metabolites are determined using infrared spectroscopy.
The concentration of a metabolite in the diagnostic sample can be expressed in a number of different ways, for example as molar concentration (number of moles of metabolite per unit volume of diagnostic sample) or mass concentration (mass of metabolite per unit volume of diagnostic sample). Alternatively, the concentration of a metabolite can be expressed as parts per million (ppm) or parts per billion (ppb). Such ways of expressing the concentration of a small molecule in solution are known in the art.
Thus, in one embodiment, the concentration of a metabolite in the diagnostic sample is the molar concentration of said metabolite. In one embodiment, the concentration of a metabolite in the diagnostic sample is the mass concentration of said metabolite.
The concentration of a metabolite in the diagnostic sample can be expressed in absolute terms, for example as absolute molar concentration or absolute mass concentration. Alternatively, the concentration of a metabolite in the diagnostic sample can be expressed by comparison to the concentration of a different metabolite in the same sample (i.e., in relative terms). By way of example, the concentration of a metabolite in the diagnostic sample in the diagnostic sample can be normalised by comparison to the concentration of a different reference metabolite within the same diagnostic sample. Examples of suitable reference metabolites are glucose and creatinine.
Step (ii) of the method as described above comprises (or consists of) comparing the concentrations in the diagnostic sample of said two or more metabolites with the concentrations of the same metabolites in at least one reference standard.
In one embodiment, a reference standard comprises (or consists of) a blood sample or samples (including a serum sample or samples and a plasma sample or samples), or a urine sample or samples, derived from blood or urine (as appropriate) obtained from a reference subject or subjects, wherein the reference subject is a subject other than the test subject.
In one embodiment, a “reference standard” comprises (or consists of) a set of data relating to the concentration of said two or more metabolites in a blood (including serum and plasma) or urine sample or samples derived from blood or urine obtained from a reference subject or subjects, wherein the reference subject is a subject other than the test subject. The set of data (as described above) is derived by measuring the concentration of said two or more metabolites. Said measuring may be carried out using any suitable technique known in the art, for example one of the following: Nuclear Magnetic Resonance (NMR) spectroscopy, mass spectrometry, gas chromatography, ultraviolet (UV) spectrometry (for example in combination with high-performance liquid chromatography [HPLC] as HPLC-UV), and infrared spectroscopy.
In one embodiment, the reference standard comprises (or consists of) a set of data relating to the concentration of said two or more metabolites in a sample or samples derived from a single reference subject, wherein the reference subject is a subject other than the test subject.
In one embodiment, the reference standard comprises (or consists of) a set of data relating to the concentration of said two or more metabolites in a sample derived from a plurality of (i.e., two or more) reference subjects. Thus, in one embodiment, the reference standard is derived by pooling data obtained from two or more (e.g., three, four, five, 10, 15, 20, or 25) reference subjects and calculating an average (for example, mean or median) concentration for each metabolite. Thus, the reference standard may reflect average concentrations of said two or more metabolites in blood and/or urine in a given population of reference subjects. Said concentrations may be expressed in absolute or relative terms, in the same manner as described above in relation to the sample that is to be tested using the method of the invention.
When comparing concentrations between the sample and the reference standard, the way in which the concentrations are expressed is matched between the sample and the reference standard. Thus, an absolute concentration can be compared with an absolute concentration, and a relative concentration can be compared with a relative concentration.
In one embodiment, the reference standard is derived from a subject (or subjects) that does not (or do not) have MS. Thus, comparison of a sample from a test subject with such a reference standard can permit the diagnosis of MS.
In one embodiment, the reference standard is derived from a subject (or subjects) having one of the following specific MS disease phases: secondary progressive, relapsing remitting, and primary progressive. Thus, in one embodiment, the reference standard is a specific MS disease phase standard.
In one embodiment, the reference standard is a secondary progressive phase MS standard. Thus, in one embodiment, the reference standard is derived from a subject or subjects previously diagnosed with secondary progressive phase MS.
In one embodiment, the reference standard is a relapsing remitting phase MS standard. Thus, in one embodiment, the reference standard is derived from a subject or subjects previously diagnosed with relapsing remitting phase MS.
The reference standard is derived from the same biological fluid type (blood or urine) as the sample that is being tested, thus allowing for an appropriate comparison between the two. Thus, by way of example, if the sample is derived from urine, the reference standard is also derived from urine. Alternatively, if the sample is a blood sample (e.g., a plasma or a serum sample), then the reference standard will also be a blood sample (e.g., a plasma sample or a serum sample, as appropriate).
Step (iii) of the method as described above comprises (or consists of) the identification of a concentration difference between the diagnostic sample and the reference standard for each of the two or more metabolites (as described above). The present inventors have discovered that variations in blood and/or urine concentrations of metabolites (as described above) can be identified in subjects having MS, as compared with healthy control subjects. Thus, said variations can be used to diagnose MS.
The term “concentration difference” embraces both positive and negative differences. Thus, a concentration difference can mean that the concentration of a metabolite is higher in the sample being tested than in the reference standard. Alternatively, a concentration difference can mean that the concentration of a metabolite is lower in the sample than in the reference standard.
The identification of a concentration difference (as described above) can be achieved using methods of statistical analysis. By way of example, NMR spectroscopy can be used to obtain an NMR spectrum for a sample. Methods of statistical analysis (for example, partial least squares discriminate analysis [PLS-DA]), can then be applied to compare said spectrum to an NMR spectrum obtained for a reference standard, allowing the identification of concentration differences.
The present inventors have discovered that variations in metabolite concentrations (as described above) can also be associated with specific MS disease phases (such as SP MS, RR MS, and PP MS). Thus, the present inventors have discovered that it is possible to diagnose a specific MS disease phase in a test subject. This can be achieved both in subjects who have not previously been diagnosed with MS and in subjects who have previously been diagnosed with MS.
Thus, in permitting the diagnosis of a disease phase of MS in a test subject, the method of the present invention provides a significant advantage over prior art methods. Prior to the present invention, diagnosis of a disease phase of MS would be achieved retrospectively, through assessment of the clinical phenotype of an MS patient. The method of the present invention allows a much more rapid diagnosis of the disease phase of MS in a patient. By way of example, the method of the present invention permits diagnosis of a disease phase of MS in the absence of outward clinical signs of said phenotype. Thus, by way of further example, the method of the present invention allows the early diagnosis of secondary progressive phase MS in a patient previously diagnosed with relapsing remitting phase MS, where the patient has yet to manifest the outward clinical signs of secondary progressive MS. The method of the present invention therefore advantageously enables medical professionals to make improved choices regarding therapy options for an MS patient, and at an earlier stage than would otherwise be possible.
The present inventors have discovered that a specific MS disease phase can be diagnosed with increased accuracy using specific subsets of the metabolites listed above.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, seven or more, or eight or more) selected from:
In one embodiment, said metabolites comprise (or consist of) the following eight blood metabolites:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
n-butyrate, N-acetyl aspartate, glucose, taurinebetaine, ascorbic acid, N-acetyl glycoprotein, fatty acid, and choline (as described above); the reference standard is derived from a subject (or subjects) that does not (or do not) have MS; and said concentration differences are selected from:
an increase in the concentration of one or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
The present inventors have discovered that changes in the concentration of the above described blood metabolites are suitable for use in diagnosing PP phase MS. Thus, in one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six) selected from:
In one embodiment, said metabolites comprise (or consist of) the following six blood metabolites:
In one embodiment, where said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, or four or more) selected from:
In one embodiment, said two or more blood metabolites further comprise a singlet (δx-y3.37) and a singlet (δx-y2.65) as measured using 1H-NMR.
In one embodiment, said metabolites comprise (or consist of) the following five blood metabolites:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a decrease in the concentration of: glucose, and phosphocholine.
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a decrease in the concentration of:
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing PP phase MS. Thus, in one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine or more) selected from:
two or more urine metabolites (for example, two, or three or more) selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, or four or more) selected from:
In one embodiment, said metabolites comprise (or consist of) the following five blood metabolites:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
a decrease in the concentration of one or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
a decrease in the concentration of:
The present inventors have discovered that changes in the concentration of the above described blood metabolites are suitable for use in diagnosing SP phase MS. In one embodiment wherein said metabolites comprise two or more blood metabolites selected from: fatty acid, ascorbic acid, n-butyrate, tyrosine and glucose (as described above); said concentration differences confirm the presence of secondary progressive (SP) phase MS.
In one embodiment, said metabolites comprise two or more (for example, two, or three or more) urine metabolites selected from:
In one embodiment, said metabolites comprise (or consist of) the following four urine metabolites:
In one embodiment, wherein said metabolites comprise two or more urine metabolites selected from:
an increase in the concentration of one or more urine metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of: TMAO, and citrate; and
a decrease in the concentration of: inositol, and lactate.
The present inventors have discovered that changes in the concentration of the above described urine metabolites are suitable for use in diagnosing SP phase MS. In one embodiment, wherein said metabolites comprise two or more urine metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, or seven or more) selected from:
In one embodiment, said metabolites comprise (or consist of) the following eight blood metabolites:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
a decrease in the concentration of one or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a decrease in the concentration of:
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing SP phase MS. In one embodiment wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six or more) selected from:
phosphocholine, lactate, N-acetyl species, and beta-hydroxybutyrate; or phosphocholine, lactate, N-acetyl species, beta-hydroxybutyrate, fatty acid, and glucose; or glucose, phosphocholine, fatty acid, lactate, alanine, beta-hydroxybutyrate, and N-acetyl species; and/or
In one embodiment, said two or more blood metabolites further comprise a singlet (δx-y3.37) as measured using 1H-NMR.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, or three or more), selected from:
In one embodiment, said metabolites comprise (or consist of) the following four blood metabolites:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
a decrease in the concentration of:
In one embodiment, said concentration differences comprise (or consist of): an increase in the concentration of a singlet (δx-y3.37) as measured using 1H-NMR; and a decrease in the concentration of:
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing SP phase MS. Thus, in one embodiment wherein said metabolites comprise two or more blood metabolites selected from:
Thus, in one embodiment wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said metabolites comprise two or more (for example, two, or three or more) urine metabolites selected from:
In one embodiment, said metabolites comprise (or consist of) the following four urine metabolites:
In one embodiment, wherein said metabolites comprise two or more urine metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
The present inventors have discovered that changes in the concentration of the above described urine metabolites are particularly suitable for use in diagnosing SP phase MS.
In one embodiment, wherein said metabolites comprise two or more urine metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six or more) selected from:
In one embodiment, said metabolites comprise (or consist of) the following seven blood metabolites:
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a decrease in the concentration of one or more blood metabolites selected from:
a decrease in the concentration of:
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing SP phase MS. In one embodiment wherein said metabolites comprise two or more blood metabolites selected from phosphocholine, N-acetyl species, and beta-hydroxybutyrate; or phosphocholine, N-acetyl species, beta-hydroxybutyrate, fatty acid, and glucose; or fatty acid, lactate, alanine, phosphocholine, beta-hydroxybutyrate, N-acetyl species, and glucose (as described above); said concentration differences confirm the presence of secondary progressive (SP) phase MS.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, or 11 or more) selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, seven or more, or eight or more) selected from:
In one embodiment, said metabolites comprise (or consist of) the following nine blood metabolites:
In one embodiment, wherein said metabolites comprise two or more metabolites selected from:
an increase in the concentration of one or more blood metabolites selected from:
a decrease in the concentration of one or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a decrease in the concentration of:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, or seven or more) selected from:
In one embodiment, said metabolites comprise (or consist of):
In one embodiment, wherein said metabolites comprise two or more metabolites selected from:
an increase in the concentration of one or more blood metabolites selected from:
a decrease in the concentration of:
In one embodiment, said concentration differences comprise (or consist of) an increase in the concentration of:
a decrease in the concentration of:
In one embodiment, wherein said metabolites comprise at least two blood metabolites selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, or seven or more) selected from:
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, or four or more) selected from:
In one embodiment, said metabolites comprise (or consist of) the following five blood metabolites:
In one embodiment, wherein said metabolites comprise two or more metabolites selected from:
a decrease in the concentration of one or more blood metabolites selected from:
an increase in the concentration of one or more blood metabolites selected from:
a decrease in the concentration of one or more blood metabolites selected from:
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a decrease in the concentration of:
In one embodiment, said concentration differences comprise (or consist of):
a decrease in the concentration of one or more blood metabolites selected from:
an increase in the concentration of:
a decrease in the concentration of: glucose, phosphocholine, and lactate.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six or more) selected from:
In one embodiment, said metabolites comprise (or consist of):
In one embodiment, wherein said metabolites comprise two or more metabolites selected from:
an increase in the concentration of one or more blood metabolites selected from:
a decrease in the concentration of:
In one embodiment, said concentration differences comprise (or consist of) an increase in the concentration of:
a decrease in the concentration of:
In one embodiment, wherein said metabolites comprise at least two blood metabolites selected from:
In one aspect, the invention provides a method for diagnosing Multiple Sclerosis in a human subject by measuring the concentration of two or more metabolites using NMR spectroscopy.
In one aspect, the invention provides an in vitro method for diagnosing MS in a human test subject using 1H-NMR spectroscopy, comprising
In one embodiment, said two or more metabolites are selected from:
blood metabolites, wherein said blood metabolites comprise:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, (δ0.90, br, CH3, δ1.30, br, CH2CH2CH3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ1.32, d, CH3, δ4.11, q, CH), and (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ1.12, m, CH); and/or
urine metabolites, wherein said urine metabolites comprise:
(δ3.26, s, ON(CH3)3), (δ2.50, d, CHAHB, δ2.65, d, CHAHB), (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said blood metabolites further comprise a singlet (δx-y3.37) and a singlet (δx-y2.65) as measured using 1H-NMR.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, or four or more) selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2CH3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2 δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2).
In one embodiment, said two or more blood metabolites further comprise a singlet (δx-y3.37) and a singlet (δx-y2.65).
In one embodiment, said metabolites comprise (or consist of) the following five blood metabolites:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (6.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2).
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ0.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2) (as described above);
the reference standard is derived from a subject (or subjects) that does not (or do not) have MS; and said concentration differences are selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), and (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2); and/or a decrease in the concentration of one or more blood metabolites selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), and (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), and (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2);
and a decrease in the concentration of:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), and (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2) and a singlet (δx-y3.37); and
a decrease in the concentration of: (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and a singlet (δx-y2.65).
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing PP phase MS. Thus, in one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2) (as described above); said concentration differences confirm the presence of primary progressive (PP) phase MS.
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ2.03, br s, COCH3), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ0.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), a singlet (δx-y3.37) and a singlet (δx-y2.65), (as described above); said concentration differences confirm the presence of primary progressive (PP) phase MS.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six or more) selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2CH3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), and (δ2.03, br s, COCH3); and/or
two or more urine metabolites (for example, two, or three or more) selected from:
(δ3.26, s, ON(CH3)3), (δ2.50, d, CHAHB, δ2.65, d, CCHAHB), (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said two or more blood metabolites further comprise a singlet (δx-y3.37).
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, or three or more), selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said metabolites comprise (or consist of) the following four blood metabolites:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H3 δ4.63, d, C(1)H), (63.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ0.32, d, CH3, δ4.11, q, CH) (as described above);
the reference standard is derived from a subject (or subjects) that does not (or do not) have MS, and said concentration differences are selected from:
a decrease in the concentration of one or more blood metabolites selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2 δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said concentration differences comprise (or consist of):
a decrease in the concentration of:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2 δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2CH3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of a singlet (δx-y3.37); and
a decrease in the concentration of:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ1.32, d, CH3, δ4.11, q, CH).
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing SP phase MS. Thus, in one embodiment wherein said metabolites comprise two or more blood metabolites selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), and (δ1.32, d, CH3, δ4.11, q, CH) (as described above);
said concentration differences confirm the presence of secondary progressive (SP) phase MS.
Thus, in one embodiment wherein said metabolites comprise two or more blood metabolites selected from:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2 δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ0.90, br, CH3, δ1.30, br, CH2CHCH2CH2C32CH3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), and a singlet (δx-y3.37) (as described above);
said concentration differences confirm the presence of secondary progressive (SP) phase MS.
In one embodiment, said metabolites comprise two or more (for example, two, or three or more) urine metabolites selected from:
(δ3.26, s, ON(CH3)3), (δ2.50, d, CHAHB, δ2.65, d, CHAHB), (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said metabolites comprise (or consist of) the following four urine metabolites:
(δ3.26, s, ON(CH3)3), (δ2.50, d, CHAHB, δ0.65, d, CHAHB), (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, wherein said metabolites comprise two or more urine metabolites selected from:
(δ3.26, s, ON(CH3)3), (δ2.50, d, CHAHB, δ2.65, d, CHAHB), (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH); the reference standard is derived from a subject (or subjects) that does not (or do not) have MS, and said concentration differences are selected from:
an increase in the concentration of one or more urine metabolites selected from: (δ3.26, s, ON(CH3)3), and (δ2.50, d, CHAHB, δ2.65, d, CHAHB); and/or
a decrease in the concentration of one or more urine metabolites selected from: (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
(δ3.26, s, ON(CH3)3), and (δ2.50, d, CHAHB, δ2.65, d, CHAHB); and
a decrease in the concentration of:
(δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
The present inventors have discovered that changes in the concentration of the above described urine metabolites are particularly suitable for use in diagnosing SP phase MS.
In one embodiment, wherein said metabolites comprise two or more urine metabolites selected from:
(δ3.26, s, ON(CH3CH3)3), (δ2.50, d, CHAHB, δ2.65, d, CHAHB), (δ3.05, s, NCH3, δ4.10, s, CH2), and (δ1.32, d, CH3, δ4.11, q, CH);
said concentration differences confirm the presence of secondary progressive (SP) phase MS.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six or more) selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H).
In one embodiment, said metabolites comprise (or consist of) the following seven blood metabolites:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H).
In one embodiment, wherein said metabolites comprise two or more blood metabolites selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ0.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H) (as described above);
the reference standard is a relapsing remitting (RR) phase MS standard, and said concentration differences are selected from:
an increase in the concentration of one or more blood metabolites selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), and (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH); and
a decrease in the concentration of one or more blood metabolites selected from:
(δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), and (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH); and
a decrease in the concentration of:
(δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H).
The present inventors have discovered that changes in the concentration of the above described blood metabolites are particularly suitable for use in diagnosing SP phase MS. In one embodiment wherein said metabolites comprise two or more blood metabolites selected from (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H) (as described above);
said concentration differences confirm the presence of secondary progressive (SP) phase MS.
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, six or more, or seven or more) selected from:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.32, d, CH3, δ4.11, q, CH), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), and (δ2.03, br s, COCH3).
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, or four or more) selected from:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2 δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ4.32, d, CH3, δ4.11, q, CH).
In one embodiment, said metabolites comprise (or consist of) the following five blood metabolites:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, wherein said metabolites comprise two or more metabolites selected from:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.32, d, CH3, δ4.11, q, CH);
the reference standard is derived from a subject (or subjects) that does not (or do not) have MS; and said concentration differences are selected from:
an increase in the concentration of one or more blood metabolites selected from:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), and (δ1.46, d, CH3; δ3.73, q, CH); and/or
a decrease in the concentration of one or more blood metabolites selected from: (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2 δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
a singlet (δx-y3.37), (δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), and (δ1.46, d, CH3; δ3.73, q, CH); and
a decrease in the concentration of:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said concentration differences comprise (or consist of):
an increase in the concentration of:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), and (δ1.46, d, CH3; δ3.73, q, CH); and
a decrease in the concentration of:
(δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), and (δ1.32, d, CH3, δ4.11, q, CH).
In one embodiment, said metabolites comprise two or more blood metabolites (for example, two, three or more, four or more, five or more, or six or more) selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H).
In one embodiment, said metabolites comprise (or consist of):
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H).
In one embodiment, wherein said metabolites comprise two or more metabolites selected from:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), (δ0.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H);
the reference standard is a secondary progressive (SP) phase MS standard, and said concentration differences are selected from:
an increase in the concentration of one or more blood metabolites selected from:
(δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H); and
a decrease in the concentration of:
(δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), and (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH).
In one embodiment, said concentration differences comprise (or consist of) an increase in the concentration of:
(δ1.46, d, CH3; δ3.73, q, CH), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ2.03, br s, COCH3), and (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H); and
a decrease in the concentration of:
(60.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.32, d, CH3, δ4.11, q, CH), and (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH).
In one embodiment, wherein said metabolites comprise at least two blood metabolites selected from:
(δ2.13, m, CH2CHNH2, δ2.45, m, OCCH2, δ3.77, t, CH2CHNH2), (δ1.46, d, CH3; δ3.73, q, CH), (δ3.42, dd, C(4)H, δ3.44, dd, C(6)HAHB, δ3.54, dd, C(2)H, δ3.73, dd, C(3)H, δ3.79, dd, C(6)HAHB, δ3.86, m, C(5)H, δ5.24, d, C(1)H) and/or (δ3.26, dd, C(2)H, δ3.40, dd, C(4)H, δ3.47, dd, C(5)H, δ3.49, dd, C(3)H, δ3.86, d, C(6)H2, δ4.63, d, C(1)H), (δ3.25, s, N(CH3)3, δ3.59, t, NCH2, δ4.17, m, NCH2CH2), (δ1.32, d, CH3, δ4.11, q, CH), (δ0.90, br, CH3, δ1.30, br, CH2CH2C3, δ1.55, br, CH2CH3, δ2.05, br, CH2CH═, δ2.25, br, CH2COOH, δ5.35, br, CH2CH═), (δ1.19, d, CH3, δ2.28, m, CHAHB, δ2.37, m, CHAHB, δ4.12, m, CH), and (δ2.03, br s, COCH3) (as described above);
said concentration differences confirm the presence of relapsing remitting (RR) phase MS.
In one embodiment wherein the method permits diagnosis of a disease phase of MS (as described above), the sample is derived from a blood sample and the reference standard is derived from blood.
In one embodiment wherein the method permits diagnosis of a disease phase of MS (as described above), the sample is derived from a serum or plasma sample and the reference standard is derived from serum or plasma.
In one embodiment, the method of the invention further comprises recording the output of at least one step on a data-storage medium. By way of example, the method of the present invention can generate data relating to the test subject, such data being recordable on a data-storage medium (for example, a form of computer memory such as a hard disk, compact disc, floppy disk, or solid state drive). Such data can comprise (or consist of) data relating to the concentration in a sample (from said test subject) of any of said two or more metabolites (as described) above.
Thus, in one aspect, the invention provides a data-storage medium comprising data obtained by the method of any preceding claim.
In one aspect, the invention provides a device for use in a method as described above, wherein said device is capable of performing the step of identifying a concentration difference for each of said two or more metabolites in the sample relative to the reference sample.
The following materials and methods were used in the examples described below.
Materials and Methods
Animal Preparation
Urine and plasma samples were obtained from a mouse model of chronic relapsing experimental autoimmune encephalomyelitis (Cr-EAE), a more clinically-relevant model for MS than the commonly used monophasic disease models. Adult Biozzi ABH mice were fed on a diet of jelly and high protein mash in the cage for a period of one week prior to Cr-EAE induction and throughout the disease time course. Cr-EAE was induced as described by Baker et al. (J Neuroimmunol. 1990; 28(3):261-70). Briefly, each animal was injected subcutaneously on day 0 and day 7 with mouse spinal cord homogenate in incomplete Freund's adjuvant supplemented with non-viable desiccated, M. tuberculosis and M. butyricum. A group of control animals was injected with the supplemented incomplete Freund's adjuvant (CFA), but omitting the spinal cord homogenate. Animals were weighed daily and assessed for clinical signs (
Blood and urine samples were taken at days 10, 14, 17, 28 and 38 after Cr-EAE induction. Blood was collected by cardiac puncture using a heparin-coated syringe and placed immediately into a blood tube containing dipotassium ethylenediaminetetraacetic acid (EDTA) on ice (Teklab, UK). Blood samples were subsequently centrifuged (14000 rpm, 5 min) at 4° C., after which the top layer of plasma was removed and stored at −80° C. Urine samples were either collected directly from the bladder after sacrificing the animal with CO2 or by placing a 96-well plate underneath the animal during sacrifice.
Human Samples
Blood serum (n=58) and urine samples (n=84) were obtained from MS patients (n=58) under Ethical approval (C03.054, Metabolic profiling of urine in MS) with cohorts of relapsing remitting (RR) (urine n=22; serum n=19), primary progressive (urine n=32; serum n=13) and secondary progressive (urine n=30; serum n=26) MS patients.
Control samples were collected from age and sex matched volunteers (urine n=28; serum n=16).
NMR Spectroscopy
Urine or blood serum/plasma samples were defrosted overnight in a cold room prior to the NMR experiments and then blood samples were further centrifuged (100000 g, 30 mins). The urine and plasma samples (100 μl EAE or 150 μl MS) were placed in a 5 mm NMR tube and diluted to a final volume of 600 μl with phosphate buffer (0.2 M Na2HPO4/0.04 NaH2PO4, pH 7.4, 0.1% sodium azide, sodium chloride 0.8%) in D2O containing 1 mM TSP (3-trimethylsilyl-1-[2,2,3,3,-2H4] propionate) as an internal standard. 1H NMR spectra were acquired from each sample using a 16.4T NMR (700 MHz 1H) system (Bruker Avance III equipped with a 1H TCI cryoprobe). For all samples a 1D NOESY presaturation sequence, with solvent pre-saturation during the relaxation delay (2 s) and mixing time (10 ms), was used. For plasma samples alone a CPMG sequence was also used with solvent presaturation and a T2 filter (total duration 40 ms; 20 loops of 2 ms spin-echoes) for suppression of lipid resonances.
Two dimensional 1H NMR spectra were acquired from a sample within each group to assist with metabolite identification. The 2D Correlation Spectroscopy (COSY) spectra were acquired on the same spectrometer as the 1D NMR spectra.
The COSY spectra were acquired with 1.5 s solvent presaturation, a spectral width of 10 ppm (7002 Hz), and 16 or 32 transients per t1 increment for 256 increments. All NMR experiments were acquired at 293 K.
The 1D 1H plasma spectra were sub-divided into 0.02 ppm regions (6=midpoint of integral region) and integrated, reducing each spectrum to 435 independent variables between 0.20-4.30 and 5.00-9.60 ppm. The integration was carried out using a macro within ACD 1D NMR Processor release 12 (Advanced Chemical Development, UK) The 1D 1H urine spectra were sub-divided into 0.02 ppm integral regions and integrated, reducing the spectrum to 385 independent variables between 0.20-4.30 and 6.00-9.60 ppm. In all spectra, the region between 4.30 and 5.00 ppm was omitted due to spectrum distortion arising from the water suppression at 4.7 ppm. In the case of the urine spectra the region between 5.00 and 6.00 ppm was also excluded to avoid the broad resonance arising from urea. These sections represent highly variable regions of the spectrum. Subsequently, statistical pattern recognition was applied to the spectra to differentiate between samples obtained from different disease states in either the EAE model or from the MS patients.
All statistical modelling was carried out using SEVICA P+12.0 (Umetrics, Sweden). All data was scaled using Pareto variance in order to suppress the noise in the data. In order to determine how predictive the models were the q2 value for each model was used as a guide to the validity of the PLS-DA model. A q2 value above 0.4 was considered statistically significant (Waterman, et al., 2010) and, therefore, tested further. The validate function within Simca P+ was used to generate 100 models where the samples were placed into random classes. The q2 values of these models 10 were then calculated and compared to the actual q2 value of the genuine model. The model was considered valid if all the predictive q2 values were lower than the real q2 value (
The models were further validated by removal of individual points within the PLS models. The models were then rebuilt and the omitted sample was then reintroduced in order to confirm that it fell within the requisite group (so-called “take one out” analysis). In order for a perfect classification the samples scored 1 if they were a member of the required group or 0 if they were not a member. An arbitrary cut-off value of 0.5 was applied to samples which did not perfectly fit the model (Brindle, et al., 2002; Gavaghan, et al., 2000). The difference between the groups was then tested with an unpaired t test to investigate the differences between the groups. If the models failed these tests they were classed as non-significant (ns), however, if they passed all the tests they were considered to be predictive models.
In order to identify the metabolites the loadings were examined by use of an s-plot, and the peaks were identified using a combination of COSY spectroscopy (
In order to confirm the identity of the singlet peaks which give no correlations within the 2D COSY spectra the predicted metabolite was added (‘spiked’) into the NMR tube as an authentic reference sample (
Cr-EAE: Animal Model
All Cr-EAE animals followed the typical disease course reported previously by Baker et al. 1990; the animals exhibited significant weight loss and increased clinical score, which peaked at day 17, and was followed by full remission such that clinical score had returned to baseline values in all animals by day 26 (
Cr-EAE: Urine Analysis
Spectra obtained by high resolution 1H NMR spectroscopy were of sufficient resolution to allow identification of over 30 metabolites. PLS analysis of the urine spectra from the naive animals at different time-points returned a model with a low q2 (0.10), indicating that it was not predictive. Consequently, all naive data were combined as a single group thereafter and analysis performed using a single control (naïve) group.
Visually, there appeared to be separation of the different disease groups, as illustrated in
The loadings from the statistically significant models generated for the disease time course identified several key metabolites that were responsible for the separations between groups (see
Cr-EAE: Plasma Analysis
Two different NMR sequences were used to obtain spectra from the plasma samples; (i) the NOESY presaturation sequence used for the urines and (ii) a CPMG sequence with a T2 lipid suppression filter. For some samples phase modulation was evident in the CPMG spectra and, therefore, the NOESY presaturation spectra were used in all subsequent analysis.
As for the urine, PLS analysis of the plasma spectra from the naive animals at different time-points returned a model with a low q2 (0.176), indicating that it was not predictive. Consequently, all naive data were combined as a single group thereafter and analysis performed using a single control (naïve) group.
Although the spectra from all groups superficially look similar upon initial overlay (
It was also possible to generate predictive models based on the spectra from the plasma of the CFA animals at all-time points during the disease course, with the exception of day 14 vs. day 28 (
On examination of the loadings within the PLS models for the Cr-EAE time course several key metabolites were identified as generating the positive separations. The initial positive models generated by comparing the different time-points for the Cr-EAE animals showed variations in n-butyrate (δx-y 0.88, 1.58) and trimethylamine N-oxide (TMAO) (δx-y 3.44). Following exclusion of these metabolites from the original spectra it was possible to generate further positive models with significant q2 values. Across all of the time point comparisons the only models that were not statistically significant were the comparisons between Cr-EAE and CFA at days 17 and 28, i.e., during Cr-EAE remission (
Key metabolites from the original models separating Cr-EAE animals from the control groups (CFA or naïve) were also identified. The metabolites that showed greatest variation in these models were: fatty acid (δx-y 0.88, 1.58, 2.03), citrate (δx-y 2.5, 2.65), lactate (δx-y 1.32, 4.11), glucose (δx-y 3.25) and phosphocholine (δx-y 3.23). As above, removal of these metabolites from the original spectra generated further positive models with significant q2 values. The metabolites that generated the positive q2 values in the second round of models were: taurine (δx-y 3.25, 3.43) and valine (δx-y 0.99, 1.04) (
All of the metabolites above showed both positive and negative changes compared to baseline over the disease time course (
In contrast to the increases observed above, the concentration of fatty acid in Cr-EAE animals was reduced during active disease, days 10, 14, and 38, relative to naive animals, but elevated during remission (day 28). At these time-points the levels of fatty acid in the CFA animals were even lower than in the Cr-EAE animals, indicating a greater effect of CFA alone than Cr-EAE on these metabolites.
No differences in TMAO were evident between Cr-EAE and naive animals. However, TMAO was lower over the early disease time course (days 10, 14, and 17) in Cr-EAE animals than CFA animals, and higher at day 38, again indicating differential effects of CFA and Cr-EAE.
Human MS: Urine Analysis
Initially, all of the MS patients were modelled against the control group. However, although positive (q2=0.11), this model did not reach significance. When the different stages of disease were considered separately against controls, all of the PLS models again returned positive q2 values indicating differences between each of these groups (RR, PP, SP) and the healthy control groups. However, only the SP vs. control model reached defined significance (q2=0.51, take-one-out analysis p<0.05;
Human MS: Serum Analysis
The same two NMR sequences as used for the animal samples were run on the human serum samples. However, unlike the spectra obtained from the animal samples, no phase modulation was evident in the spectra obtained from the human samples. Since there were no differences between the two types of spectra, the CPMG spectra were used in all subsequent analysis. The high resolution 1H NMR spectroscopy furnished spectra of sufficient resolution to identify over 30 metabolites (
Once again the whole cohort of MS patients was compared to the healthy control group. This model showed a significant separation between the two groups with a q2 value of 0.40; take-one-out analysis p<0.05. Subsequent to this, the different disease states were considered individually. All of the individual comparisons between the MS patient groups (RR, PP, SP) and the healthy control group (
Following the initial analysis, the metabolites responsible in each group for causing separations were removed from the spectra of that particular group and a second 19 round of positive models were generated (
Comparison of the RR and SP groups generated a significant model, with a q2 value of 0.46. Subsequent take-one-out analysis generated a box and whisker plot which showed a statistically significant difference between the two groups with a p value <0.0001 (
In the case of the RR vs. SP model, after the first round of analysis fatty acid (δx-y 0.88, 1.58 1.3) and lactate (δx-y 1.32) were found to be higher in the SP group, whilst alanine (δx-y 3.73) and phosphocholine (δx-y 3.23) were found to be lower in the SP group than the RR group (
Subjects/Materials and Methods
Human Samples
Samples from three cohorts of MS patients as well as age- and sex-matched control volunteers were obtained with ethical approval (UK NHS C03.054, Metabolic profiling of urine in MS and 08/H0604/155): Set A: urine n=22, 30 and 28, serum n=15, 38 and 10; Set B: serum n=6, 10 and 7; Set C: serum n=5, 10 and 7 in RRMS, SPMS and control volunteers respectively. Set A urine and serum samples were also collected for primary progressive (PP) MS patients (n=32 and 13 respectively). Serum samples were collected by centrifugation (3000×g, 10 mins) after allowing blood to clot for 30 min at room temperature in Vacutainers containing clot activator and gel (BD, UK). Urine samples were collected mid-stream in pots without additives. Serum and urine were aliquoted and stored at −80° C. Clinical details of MS patients are given in
NMR Spectroscopy
Samples were defrosted at 4° C. prior to NMR. Blood samples were further centrifuged (100,000×g, 30 min, 4° C.). Urine and plasma (100 μl EAE or 150 μl MS) were diluted in a 5 mm NMR tube to a final volume of 600 μL with 0.24M sodium phosphate buffer (pH 7.4, 0.1% NaN3, 0.8% NaCl) in D2O containing 1 mM TSP (3-trimethylsilyl-1-[2,2,3,3,-2H4] propionate) as an internal standard. 1H NMR spectra were acquired from each sample using a 16.4T NMR (700 MHz 1H) system (Bruker Avance III equipped with a 1H TCI cryoprobe). For all samples a 1D NOESY pre-saturation sequence, with solvent pre-saturation during the relaxation delay (2s) and mixing time (10 ms), was used. For blood samples alone a CPMG sequence was also used with solvent pre-saturation (2s) and a T2 filter (total duration 40 ms; 20 loops of 2 ms spin-echoes) for suppression of lipid resonances. All 1D spectra were automatically baseline corrected using a 0th order polynomial (Topspin 3.0). Two-dimensional Correlation Spectroscopy (COSY)1H NMR spectra were acquired from a sample within each group to assist with metabolite identification. The COSY spectra were acquired with 1.5 s solvent presaturation, a spectral width of 10 ppm (7002 Hz), and 16 or 32 transients per t1 increment for 256 increments. All NMR spectra were acquired at 293K.
Data Analysis
Non-linear peak alignment16 was performed on each 1D 1H spectra which were then subdivided into 0.02 ppm regions (6=midpoint of integral region) and integrated between 0.20-9.60 ppm using a custom MATLAB script. In all spectra, the highly variable region between 4.30-5.00 ppm arising due to water suppression was excluded. In urine spectra, the variable urea resonance between 5.00-6.00 ppm was also excluded. Thus spectra were reduced to 435 (blood) or 385 (urine) independent variables. Data were scaled using the Pareto variance to suppress noise and PLS/DA was applied. Integral buckets containing gross contaminants (e.g., organic solvent, EDTA) were excluded from analysis.
Statistical Methods
For each comparison, a PLS-DA model was derived which best explained the differences between the variables for the groups being studied (SIMCA P+12.0, Umetrics, Sweden). To determine the potential predictive nature of the models, the q2 value was calculated, q2 is derived from a step-wise cross-validation of the model whereby a model generated with a subset of samples removed is used to predict group membership of the missing samples. A value of q2>0 means the model is predictive. It is generally held that q2>0.4 is the threshold for significance for biological modelling17.
In addition, model validation was carried out using a pseudo-Monte Carlo method where 100 models were built using random group assignments. Only models where the genuine q2 was higher than 95% of the randomly generated q2 values were considered significant (
In order to identify the metabolites underlying group separations, the variable loadings of each model were examined and the relevant resonances identified using a combination of COSY spectroscopy (
To validate the models generated additional sample sets (Sets B and C) were used. PPMS patients were not included owing to lack of samples. Set B was used to build predictive models and Set C was used as a testing and validation set i.e., Set C were blinded and the models built using Set B were used to predict group membership for each sample in Set C. The results are expressed as a 2×2 contingency table for each model. Fisher's exact statistic was calculated in each case. Receiver operator characteristic (ROC) curves were constructed for each model and the area under the curve determined. Model membership probability thresholds derived from the model-building set were used to determine sensitivity and specificity in the prediction set (Set C).
Results
Initial MS Sample Set (Set A)
We began with analysis of serum samples from MS patients in Set A. A model built using the CPMG spectra and separating all MS patients from the control cohort was predictive (q2=0.41). Furthermore, the comparisons between individual MS patient and control groups were significant (q2=0.70, 0.61 and 0.42 for control vs. PPMS, RRMS and SPMS respectively;
When making comparisons between MS groups, the model comparing the RR and SP groups revealed a significant separation (q2=0.45;
The urine analysis produced models which were not as strong as those built from the serum samples. Models constructed comparing control volunteer urine to each of the MS groups were all predictive but only control vs. SPMS reached the defined significance of q2>0.4 (Control vs. RRMS q2=0.22, vs. PPMS q2=0.20, vs. SPMS q2=0.51). For the intra-MS urine comparisons, both PPMS vs. RRMS and PPMS vs. SPMS models proved to be non-predictive whilst SPMS vs. RRMS proved predictive but non-significant (q2=0.33;
Independent MS Sample Set Analysis (Sets B and C)
To validate the models generated from sample Set A, two independent sets of serum samples were used, Sets B and C. Samples from Set B were used in the same manner as the samples from Set A and three new models were generated: ControlB vs. RRMSB, ControlB vs. SPMSB and RRMSB vs. SPMSB. In each case, the models from Set B were predictive and were all validated in the same manner as the models from Set A. As previously, the q2 values were greater than 0.4 for each model: ControlB vs. RRMSB, q2=0.62; ControlB vs. SPMSB, q2=0.77; and RRMSBVS. SPMSB, q2=0.48. Moreover, upon examination of the loadings, the same metabolites were found to be responsible for the separations observed between groups in this secondary dataset with the exception of glucose, which was not as important in separating SPMS from RRMS patients in the secondary data set. This is likely to be owing to variation in sample handling technique in the clinic e.g., time of clinic or duration since last meal.
Each new model generated from Set B was tested for its predictive ability using Set C, the second independent data set which had not been used in building any models. All Set B models were both sensitive and specific at predicting group membership for patients: RRMS vs. SPMS model, sensitivity=0.9 and specificity=0.8; RRMS and SPMS vs. control models, sensitivity and specificity=1.0. Contingency tables showing correct and incorrect classifications for each model are given in
Fan W M T. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy. 1996; 28:161-219. Gavaghan C L, Holmes E, Lenz E, Wilson I D, Nicholson J K. An NMR-based metabonomic approach to investigate the biochemical consequences of genetic strain differences: application to the C57BL10J and Alpk: ApfCD mouse. FEBS letters. 2000; 484(3): 169-74.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1202092.1 | Feb 2012 | GB | national |
This patent application is a continuation of U.S. patent application Ser. No. 14/377,454, filed Aug. 7, 2014, now abandoned, which is the National Stage of International Application No. PCT/GB2013/050285, filed Feb. 7, 2013, which claims priority to GB 1202092.1, filed Feb. 7, 2012, the disclosures of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14377454 | Aug 2014 | US |
| Child | 15963271 | US |
