WO2013119651A1

WO2013119651A1 - Methods for diagnosing cognitive impairment

Info

Publication number: WO2013119651A1
Application number: PCT/US2013/024916
Authority: WO
Inventors: Ziad S. RAMADAN; Steven S. Hannah; Brian M. ZANGHI
Original assignee: Nestec Sa
Priority date: 2012-02-08
Filing date: 2013-02-06
Publication date: 2013-08-15

Abstract

The invention provides methods for diagnosing cognitive impairment in an animal. The methods comprise obtaining a sample from the animal; analyzing the sample for the presence of one or more metabolites associated with cognitive impairment; comparing the amount of metabolites identified in the sample to a corresponding amount of the same metabolites present in a sample from one or more comparable control animals that do not suffer from cognitive impairment; and using said comparison to diagnose cognitive impairment in the animal if the metabolites found in the animal's sample is greater or less than the amount present in the control sample.

Description

METHODS FOR DIAGNOSING COGNITIVE- IMPAIRMENT

GROSS REFERENCE TO RELATED APPLICATIONS

[000.1 j This application claims priority to U.S. Provisional Application No. 61/596402 fifed February 8, 2012. the disclosure of which is incorporated herein by this reference.

BACKGROUND OF THE INVENTION

Field of the Invention

|0002| The invention relates generally to methods for diagnosing and predicting cognitive impairment and particularly to methods for diagnosing and predicting cognitive impairment by measuring metabolites associated with cognitive impairment

Description of Related Art

(0003J Aged or aging animals frequently suffer some degree of cognitive impairment. Canine Cognitive Dysfunction Syndrome (CCDS) is the age related deterioration of cognitive abilities. Dogs with CCDS exhibit various behavioral, disorders, for example, they may not respond to their name or familiar commands, may get lost or confused even in familiar surroundings, may no longer greet or respond to their owners or visitors, may exhibit diminished daytime activity, may walk in circles, may shun affection, and may lose bladder or bowel control. Early detection of cognitive impairment will help healthcare professionals and pet owners make informed choices about reducing the risk of developing CDS in their dogs. Nutritional supplementation, has a potentially valuable role to play in maximizing the benefit of therapy in terms of increasing the animal's quality of life. Key to this benefit is the early detection of cognitive impairment thereby- enabling animals to receive the most appropriate care,

[0004] Biomarkers are useful for detecting conditions when an animal is displaying minimal symptoms or asymptomatic. Metabolites are useful biomarkers. Currently there are no known biomarkers useful as diagnostic agents to .measure cognitive impairment in animals.

(0005] Therefore there remains a need for diagnosing and predicting cognitive impairment in animals to provide the most appropriate and effective level of treatment. Such treatment will improve the animal's quality of life. The present invention satisfies this need.

SUMMARY OF THE INVENTION

[0006] It is, therefore, an object of the present invention to provide methods for diagnosing and predicting cognitive impairment in animals.

! [Ό007] This and other objects are achieved using methods for diagnosing and predicting cognitive impairment in an animal that involve obtaining a [serum] sample from the animal; analyzing the sample for the presence of one or more metabolites associated with cognitive impairment; comparing the amount of metabolites identified in the sample to a corresponding amount of the same metabolites present in a sample from one or more comparable control animals that do not suffer from cognitive impairment; and using said comparison to diagnose cognitive impairment in the animal if the metabolites found in the animal's sample is greater or less than the amount present in the control sample.

[0008] Other and further objects, features, and advantages of the present invention will be readi ly apparent to those ski lled in the art.

DETAILED DESCRIPTION OF THE IN VENTION

Definitions

[0009] The term "animal" means any animal susceptible to or suffering from cognitive impairment, including human, avian, bovine, canine, equine, feline, hicrine, lupine, murine, ovine_* or porcine animals.

[0010] By the term "metabolite", it is meant specific small molecules, the levels or intensities of which are measured in a sample, and that may be used as markers to diagnose a disease state. These small molecules may also be referred to herein as "metabolite marker'; "metabolite component^'5, "biomarker", or ''biochemical marker,^"

[^'0011] The term "comparable control animal" means an animal of the same species and type or an individual animal evaluated at two different times.

[0012j The term "companion animals" means domesticated animals such as dogs, eats, birds, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, pleasure horses, cows, goals, sheep, donkeys, pigs, and more exotic species kept by humans for company, amusement, psychological support, extrovert display, and all of the other functions that humans need to share with animals of other species.

{00131 The terra "diagnosing" means determining if an animal is suffering from or predicting if the animal is susceptible to developing cognitive impairment.

[0014] As used herein, ranges are used herein in shorthand, so as to avoid having to list and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. f flOI S] As used herein, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references "a", "an", and "the" are generally inclusive of the plurals of the respective terms. For example, reference to '"a method" includes a plurality of such "methods.'" Similarly, the words "comprise", "comprises", and ''comprising" are to be interpreted inclusively rather than exclusively. Likewise the terras "include", "including" and "or" should all be construed to be inclusive, unless such a construction is clearly prohibited from the context.

[001.6] The methods and compositions and other advances disclosed here are not limited to particular methodology, protocols, and reagents described herein because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to, and does not, limit the scope of that which is disclosed or claimed.

[0017} Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the fiekl(s) of the invention, or in the fie!d(s) where the term is used.

(0018) All patents, patent applications, publications, technical and/or scholarly articles, and other references cited or referred to herein are in their entirety incorporated herein by reference to the extent allowed by law. The discussion of those references is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, are relevant, material, or prior art. The right to challenge the accuracy and pertinence of any assertion of such patents, patent applications, publications, and other references as relevant, materia}, or prior art is specifically reserved.

The invention

[0019] In one aspect, the invention provides methods for diagnosing cognitive impairment in an animal. The methods comprise obtaining a sample from the animal; analyzing the sample for the presence of one or more metabolites associated with, cognitive impairment: comparing the amount of metabolites identified in the sample to a corresponding amount of the same metabolites present in a sample from one or more comparable coniro! animais that do not suffer from cognitive impairment; and using said comparison to diagnose cognitive impairment in the animal if the .metabolites found in the animal's sample is greater or less than the amount present in the control sample. (0020] In another aspect, the invention provides methods for determining if an animal is responding to treatment for cognitive impairment. The methods comprise obtaining a sample from the animal; analyzing the sample for the presence of one or more metabolites associated with cognitive impairment; comparing the amount of metabolites identified in the sample to a corresponding amount of the same metabolites present in a previous sample from the same animal; and using said comparison to determine an improvement in the animal if the metabolites found in the animal's sample are lower or higher the amount present in the previous sample. (0021] The inventions is based upon the discovery that the metabolites of the invention are present in the sample of an animal and that the amount of the metabolites in the sample serves as a biochemical indicator for diagnosing cognitive impairment by indicating or predicting the threshold for cognitive impairment. The invention allows veterinary and other clinicians to perform tests for these "biomarkers" in a sample and determine whether the animal is susceptible to or suffering from cognitive impairment and whether there is a need for further diagnostics or treatment, Having established the need for further diagnostics or treatments, the cost and risk of such further diagnostics or treatments are justified.

[0022} In various embodiments, one or more comparable control animals that are not the animal being evaluated for cognitive impairment and that have been determined not to suffer from cognitive impairment are evaluated for at least one of the metabolites and the results of such evaluations are used as a baseline value for comparison with the results from an animal being evaluated for one or more of the metabolites. In preferred embodiments, the baseline value for the metabolites is determined by evaluating numerous comparable control animals.

[0023] In another, the amount of at least one of the metabolites are determined for an animal at various time throughout the animal^'s life and the results used to determine if the animal is susceptible to or suffering from cognitive impairment, e.g. , if the amount of at least one of the metabolites increases or decreases as the animal ages, the animal can be diagnosed as susceptible to or suffering from cognitive impairment. In preferred embodiments, the animal is evaluated periodically and the results for the metabolites are recorded. Then, if a subsequent evaluation shows that the amount of one or more metabolites has increased or decreased since the last evaluation(s). the animal is diagnosed as susceptible to or suffering from cognitive impairment.

[0024} Any sample that is of biological origin may be useful i the present invention. Examples include, but are not limited to, blood (serum/plasma), cerebral spinal fluid (CSF), urine, stool, breath, saliva, or biopsy of any tissue, In one embodiment, the sample is a serum sample. While the term "serum^" is used herein, those skilled in the art will recognize that plasma or whole blood or a sub-fraction of whole blood may also be used, in one embodiment, the sample is a serum sample.

[0025] The step of analyzing may comprise analyzing the sample by liquid chromatography mass spectrometry (LC-MS), or alternatively may comprise analyzing the sample by liquid chromatography and linear ion trap mass spectrometry when the method is a high-throughput method.

[0026] The extracted samples may be analyzed using any suitable method know in the art. For example, and without wishing io be limiting in any manner, extracts of biological samples are amenable to analysis on essentially any mass spectrometry platform, either by direct injection or following chromatographic separation. Typical mass spectrometers are comprised of a source which ionizes molecules within the sample, and a detector for detecting the ionized .molecules or fragments of molecules. Non-limiting examples of common sources include electron impact, electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photo ionization (APP!), matrix assisted laser desofption ionization (MALDI), surface enhanced laser desorption ionization (SELDl), and derivations thereof. Common, mass separation and detection, systems can include quadrupole, quadrupole ion trap, linear ion trap, lime-of-ilighi (TOF). magnetic sector, ion cyclotron (FTMS), Orbi rap, and derivations and combinations thereof. The advantage of FTMS over other MS- based platforms is its high resolving capability that allows for the separation of metabolites differing by only hundredths of a Dalton, man which would be missed by lower resolution instruments.

[0027] The metabolites are generally characterized by their accurate mass, as measured by mass spectrometry technique used in the above method. The accurate mass may also be referred, to as "accurate neutral m ss" or "neutral mass". The accurate mass of a metabolite is given herein in Daltons (Da), or a mass substantially equivalent thereto. By "substantially equivalent thereto", it is meant thai a +/-S ppm difference in the accurate mass would indicate the same metabolite, as would be recognized by a person of skill in the art. The accurate mass is given as the mass of the neutral metabolite. As would be recognized by a person of skill in the art, the ionization of the metabolites, which occurs during analysis of the sample, the metabolite will cause either a loss or gain of one or more hydrogen atoms and a loss or gain of an electron. This changes the accurate mass to the "ionized mass", which differs from the accurate mass by the mass of hydrogens and electrons lost or gained during ionization. Unless otherwise specified, the accurate neutral mass will be referred to herein.

10028] Similarly, when a metabolite is described by its molecular formula or structure, the molecular formula or structure of the neutral metabolite will be given. Naturally, the molecular formula or structure of the ionized metabolite will differ from the neutral molecular formula or structure by the number of hydrogens lost or gained during ionization.

[0029 [ While the use of one of the metabolites is sufficient for diagnosing cognitive impairment; the use of two or more of such metabolites is encompassed within the in vention and may be preferred in many circumstances. The metabolites can be evaluated and used for a diagnosis in any combination.

[0030] in one embodiment, the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 431.3845, 586.4545, 428,366, 429.3692, 859.9152, 783.5214, 600.47. 430.3815, 746.6425, 918.5546, 230,8621 , 476.3499, 300.266, 691.5464, 700.4985, 743.5836, 766.4501 , 801.0394, 1033.776, 658.4797, 690.5431, 920.7392, 762.5803, 659,484, 620.5556, 603.5657, 189.0784, 212.1 1 15, 304.6394, 1085.962, 1056,984, 725.5073, 634.289, 755.4417, 549.623, 61 1 ,2828, 621.2718, 831.6701 , 264.1004, 237.8986, 733.477, 193.883, 237.5638, 577.251 1 , 610.2803, 712.4157, 431 .385, 678.278, 437.913, 612.553, 445,363, 287.77, 808.661, 272.751, 291.931, 312.23, 686.235, 606.257, 291.596, 607.261, 658.48, 301.949, 653.267, 652.264.

{0031| In one embodiment, the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalen to, 431 .3845, 586.4545. 428.366, 429.3692, 859.9152, 783.5214, 600.47, 430.3815, 746.6425, 918.5546, 230.8621 , 476.3499, 300.266, 691 ,5464, 700.4985, 743.5836, 766.4501 , 801 ,0394 , 1033.776, 658.4797, 690.5431 , 920.7392, 762,5803, 659.484, and 620,5556.

|0032] In one embodiment, the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 603.5657, 189.0784, 212.1 1 15, 304.6394, 1085.962, 230.8621 , 1056.984, 428,366, 725.5073, 586.4545, 634.289, 755.4417, 620.5556, 549,623, 600.47, 611.2828, 621.2718, 746.6425, 831.6701, 264.1004, 783.521.4, 237,8986, 691.5464, 429.3692, 733.477, 193.883, 237.5638, 577.251 1 , 610.2803. and 712.4 ( 57. [0033] In one embodiment, the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 431.385, 678,278, 437.913, 612.553, 445.363, 287,77, 808.661 , 272.751 , 291.931,. 659.484, 312,23, 686.235, 606,257, 291.596, 607,261 , 658.48, 301 ,949, 653,267, and 652.264.

[0034] In one embodiment, the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 430,3815. 428,366, 783,5214, 600.47, 746.6425, 918.5.546, 230.8621 , 476.3499, and 300,266,

[0035] In various embodiments, the animal is a human or companion animal. Preferably, the companion animal is a canine such as a canine such as a dog or a feline such as a cat.

EXAMPLES

[0036| The invention can be further illustrated by the following examples, although it will be understood that these examples are included merely for purposes of illustration and are not intended to limit the scope of the invention unless otherwise specifical l y indicated,

[0037] Study Design. Sixty subjects were selected from a pool of seventy dogs, which were at least eight, years of age. The selection was based on memory performance using a variable delay non-matching to position task (varDNMP First, the dogs were placed in three non-overlapping groups that were defined, as:

Group A: High Performing Memory (best performing group), N-12

Group B: Low Performing Memory (most poorly performing group), N-24

Group C: Moderate Performing Memory (intermediate in performance), N-24

[00381 DNMP testing was carried out at baseline and again after 45 days. Dogs were tested on three-choice varDNMP for 10 baseline days, and for 14 days during retest. All DNMP testing will consist of variable-delayed testing in which delays of 20 and 90 seconds will occur with equal frequency over the 12 test trials per day, resulting in 60 trials for each delay during baseline and 84 trials for each dela during retest. The delays will occur randomly within the test sessions, with the constraint that each delay will occur on exactly 6 trials.

[0039] Serum samples were collected at 8AM from all 60 dogs at baseline. Serum samples from 36 dogs were analyzed when collected 70 days later. Specifically, all 12 HMP dogs, 12 MMP dogs, and 12 LMP dogs. Serum samples were stored at -80 degrees Celsius until the date of extraction, after which the extracts were stored at -80 degrees Celsius until the date of analysis. For each sample, 400 uL of serum was extracted according to Phenomenome

Discoveries Inc. SOP# VAL-053-OL

(0040] Mass ^'Spectrometry Analysis. The prepared extracts were analyzed using Phenomenome Discoveries Inc. SOP# PR.GT-017-06 for Fourier Transform Mass Spectrometry (FTMS) analysis using the following modes; electrospray ionization {^'positive and negative) and atmospheric pressure chemical ionization (positive and negative), each with organic and aqueous phases of the extraction. Collectively, six separate injections were performed on each sample. The FTMS analysis resulted in. highly resolved mass-to-charge ratios,

[004.1] Mass Spectrometry Data Analysis. Each complex spectrum was processed in the following manner, according to Phenomenome Discoveries Inc. SOP# PROT-021 ~03: internal mass axis calibration: Each mode of FTMS analysis was calibrated to ensure ultra-high mass accuracy prior to the peak selection process. All errors are less than or equal to 1 ppm. Generally the error is between 0.001 ppm and 0.1 ppm. Peak selection: For each analysis mode, a combination of automated and manual peak, picking processes were employed to ensure that only peaks that represent real metabolites were selected for further processing. Peak processing: Upon completion of this stage of data analysis, confirmed and selected peak lists for each mode were processed into individual mode-specific DiSCO^'VAmetrics™ (Phenomenome Discoveries inc., Saskatoon, SK, Canada) files using mode-specific algorithms.

[0042) Primary mode-specific DlSCOVAmetrics™ files were combined resulting in one final DISCOVAmetrics™ file per sample; this file represented the data from all modes of DlSCOVAmetrics™ tiles collected. The files generated contain the neutral mass of all of the 12C and higher intensity 13C metabolites. The intensities were expressed as a signal-to-noise fS N) ratio. It also includes calibration statistics for validation purposes.

Example i

[00431 Analysis using al l samples (baseline and end~of«study). The comparison of the 3 groups using the complete data set, including both sample collections (baseline and end) identified 777 masses with p-value s less than 0.05. P -tests between the three groups of animals using all of the samples identified 103 masses with p-value less than 0.005, The primary component analysis (PCA) created using these masses identified a subset of LMP dogs that separated from the other dogs. Thirteen masses were found to have a p-value less than 0.0.0 . The most statistically significant metabolic changes detected in the comparison of the three memory performance groups are shown below in Table L The table includes the mass, p- value, false discovery rate (FDR), putative formula assignment, putative metabolite identification, and the ratio of the average signal to noise (S/N) across the groups. The average S/N (·.*.· standard error) was calculated for the mass across all samples collected from the group.

Table 1

M ss p-vaJue FDR Formula Metabolite BMP LMP MMP

431.3845 3.72E-05 0,18543062 C13 1.8 0.2 3.U0. ! .6■{}. !

586.4545 S.83E-05 0.14557791 1.6*0.2 30:0.3 E7HU

428.366 6.06E-05 0.10088508 C29H4802 1 -Hydroxy 26.5±2 42. 4 25.7±1.4 stigmast-4-ert-3- one

429.3692 \ MM Art 0.13767008 Ci3 82-0.6 13.M4.3 8.0*0.4

S59.9t.52 1.40E-04 0. S3 81369 C13 3. &;.(.».?. 3.5 0. i 3.E-HU

783.5214 I.72E-04 0.14314065 C46M74NO l-&lkc-nyk2~ 4. it 0.3 2.8*0.2 39 :0.2

?P acyi-PliP-M- MeE

600.47 2.46E-04 0.17554553 38 >0.3 2.9H) 3 1.6*0.1

430.3815 2.65E-04 0.1 532 Ϊ6 C29HS0O2 alpha- 5."._:.0.4 9.3; U 5.1*0.5

Tocopherol

746.6425 3.36B-04 0.18617441 C47H8606 Triacyigiyceroi 13,8*0.4 ii.8r(54 ί 3.1 0,2

9 i 8.5546 6.! 51 -04 0.3070! 528 Novel 6.910.4 9.5-J--06 6.9*0.5

Metabolite

230.862! 6.28E-04 0.28505878 Novel 2.6i0.2 3.0._:.0.2 2.3±0.1

Metabolite

476.3499 6.S2E-04 0,27095949 C29H4805 2,3.22,23- 4.8.0.3 6.3 '0.4 4.6x0.3

Tetrahydroxysti

gmast-24(28)- en-6~ ne

.300.266 9.37E-04 0.3596845 ! C18H3 03 16- 4.4 0.1 S.(H0.2 4.7.0.2

Hydroxyoctadec

anoie acid

691.5464 0.00! 0.35706053 Ci.3 1.3±L4 35.9÷.1.3 29,9÷-0,9

700.498.5 0.00! 0.33897945 CI3 5.0 0.5 2. 0. 4.3*0.4

743.5836 0.001 0.3370957 C42MS2NO l-a!kyi-2-ae l- 33,6i.:Li 2S.4±LS 34.1:1:1.3

7.P sn-glycero-3-

PIsPC

766.4501 0.001 0.31.8279 1 C IH6601 2.2*0.1 1.7 0. ί 7.2 ' 0 ί

3 3,22,24-triol

80 i .0394 0.00 ϊί 0,315000! 8 Novel 6.1 : .2 6.2d:0.2 7.2*0.2

Metabolite Mass p-value FDR Formula Metabolite HMP I MP M ^'P

1033.776 0.0 13 0,33074566 C 13 2 3-> 0. i 3.3*0.3 2, 3+0.2

658.4797 0,003.3 0.3184805 C40B66O7 2.5*0,4 4.4x0.5 2.3*0.3

690 543 ! 0.0013 0.31 S77SI C42R7407 66.4.±2.9 76. 1*2 ? 64.0.3:2

920.7392 0.0014 0 3073023 C66H9602 6_*6*0.8 1 1.8* 1 .9 5.0*0.8

762.5803 0,0014 0.2960087 C49H7806 Triacy!g!ycerol 2.0+0,2 3.5*0.4 2. $±0.2

659,484 0,00 ! 5 0.30295983 C L 1.5*0.1 2.2+-0.2 1.6*0. !

620.55S6 0.00 ! 5 0.29097247 C43H7202 5.7+0.3 4.5 vi) 2 5.6*0.2

|^' 04 ] Referring to Table .1 , the data show the most statistically significant metabolic changes detected in the comparison of the three memory pedbrmance groups (including both baseline and end of treatment samples).

Example 2

{^"6045] Analysis using baseline samples. The comparisons of the three animal groups were repeated using the .first collection samples (baseline) alone. The group comparison using the baseline samples identified 358 masses with p<0.05. The PCA created using the masses with p<0.05 showed a separation of a small subset of the LMP dogs. When a more stringent cut-off was utilised the list of statistically significant masses was reduced to 30 (p<0,00S). The most statistically significant masses are listed in Table 2 below along with the putative metabolite identifications.

Table 2

Mass value FOR Formula Metabolite BMP L.MP MMP

577.25 H 0.004698 0.83736 ! 42 aromatic 20. 0.ό ! 6.7^0.8 1.7.8+0.

62 S hydrocarbons with 5

amide and thio

6I0.2S03 0.004933 0.84896343 C_¾H_¾N,O_s 235.7±6, 1 2. ! -i-S, 204.516

86 P 9 3 .2

712. 157 0.00-1936 0,82105341 10+0.4 S.Si;0.3 9,7±0,3

19

[0( 46] Referring to Table 2. the data show the most statistically significant metabolic changes detected in the comparison of the three memory performance groups (only baseline samples). The Table includes the mass, p-value, false discovery rate (FDR), putative formula assignment, putative metabolite identification, and the ratio of the average signal to noise (S N) across the groups. The average S/N (± standard error) was calculated for the mass across all samples collected from the group.

Example 3

[0047] Analysis using the end-of-study samples. The analysis of the three groups of animals using the second collection (end-of-study) samples, identified 389 masses with p<0.05. The PCA created using the masses with p<0.05 showed a separation of a small subset of the L.MP dogs. Approximately 65% of the variance was captured across the first three principal, components. When a more stringent cut-off was utilized the list of statistically significant masses was reduced to 20 (p ().005).

[0048] The most statistically significant masses are listed in Table 3 along with the putative metabolite identifications. The table includes the mass, p- value, false discovery rate (FDR), the analysis mode the mass was detected in, putative formula, assignment, putative metabolite identification, and the ratio of the average signal to noise (S/N) across the groups. The average S/N (.*. standard error) was calculated .tor the mass across all samples collected from the group. Elevated levels of putativeiy identified 1 5,16-DiHOD.E, an oxygenated lipid found in blood, was detected in the IMP animals. Many of the other masses in this table have been putativeiy assigned formulas mat contain sulfur. There were also a few masses that we were unable to assign putative formulas to, and may therefore be novel metabolites. Many of the masses were detected at. elevated levels in the LMP animals in comparison to the other groups.

Table 3

Mass p-value FDR Formula Metabolite ! ttPM LPM MP^'M Mass p-vaiue FDR Formula Metabolite HPM I.I'M Mi'.M

431.385 7.2 E-04 3.60586S02 C13 Alpha L 91-0.2 3.5+0.6 i.41:0.1

Tocopherol

678,278 7.29E-G4 LSI 926 14 C_¾H₄-_fO_?PS 2.9+02 1.8+0.1 2.7+0,2

437.913 9.80E-04 1,63071063 2+0.2 2.9+0.2 2+0.1

S

612.553 0.00! 10436 1,37769042 C_¾!¾O_sS 1.5 + 0.2 3.4+0.6 L710.2

445.363 0,00114979 1.14749206 ^'Novei 2.7+06 5.3+1 1.4+0,3

Metabolite

287.77 0.00116733 0,97083128 Novel 33.10.3 5.3+0.5 3.2+0,3

Metabolite

808.661 0.00143162 1,02053851 C₄₉K_nQ_&$ 2.4+0.2 3.7+0,4 2,6.-|-0. ί

272.75! 00 15 168 0.9553884? Move! 4+0.2 3.1+0.2 4.4+0.2

Metabolite

291 ,931 0.00573043 0.95942527 C₆H,O_;P_:>K. 4.8+0.5 7.6+0.6 51.0.5

659.484 0.00231053 1,1.529569 C₃₄H_7i O,P 1.9+0.2 2.8+0.3 !.7_.-04

Ma

312.23 0,00246109 1.11643768 15,16 2.510.2 3.5+0.3 2.5+0.2

DsHODE

686.235 0.00259567 1.07936597 C,_; ¾i>_;8 3.6+0.9 6.S + I.1 1.6+0.4

606.25? 0.00296398 1,13771236 C: 4 :(.>:; 453.7+27.2 360.3+21. 479.3+2

5

291.596 0.00343714 1.2250937 Novel 7.7+0,? 11.310.9 8+0.7 etaboiite

607.261 0.0035464 1,1797677 S 163.3+9,6 130.8+7.8 ί 72.4+7

.8

658.48 0.00391301 1.22036894 ,S!,„0. TAGf20::5/15 3.6+0.6 6.2+0,8 2.8+0.6

:0(OH)/Ac)

301.949 0.00428712 1.25839724 Novei 7.9+0,5 S 0.5.1-0.7 7.9+0,4

Metabolite

653.267 0.00443644 1.22987895 1 1.6+9.2 112.2+7.7 L i + 7

652.264 0.00492778 941S959 C,,H,_t)0,S 379.9+24.8 299.8120. 404. + 1

9 9,5 f^'0049] Referring to Table 3, ihe data show the most statistically significant metabolic- changes detected in the comparison of ihe samples collected the second time (end) from groups A. B and

<::.

Example 4

[0050j Baseline versus End-of-stody, There vvere 48 masses found in common between the statistically significant (p<0.05) masses in the first and second sample collections, 9 of which were also found to be amongst the most statistically significant masses (p<0.001) when all samples were analyzed together. The majority of the masses (? out of 9) were detected at increased levels in the IMP dogs as compared to the other two groups. The putative metabolite identifications of these masses are provided in Table 4.

Table 4

(^'0051] Referring to Table 4, the data show the putative metabolite identifications of the 9 masses found statistically significant in the comparison of the three groups of animals in all the samples.

(0052) These results show that lipid metabolism has been altered in the IMP dogs, as increased levels of steroids and decreased glycerolipids are detected. The increased levels of choiesteryl acetate and - tocopherol (vitamin E) in the LMP dogs are the most significant metabolic differences between the groups of animals, detected in both sample collections (baseline and end). Profile matching using a Pearson correlation was used to find other masses with similar profiles to that of choiesteryl acetate and a-tocopheroL

0053} The most robust metabolic differences were observed in the LMP (memory demented) dogs in comparison to the other two groups of dogs. A subset of the LMP dogs appeared to have significantly altered lipid metabolism, as evidenced by elevated levels of sterol metabolites (choiesteryl acetate, stigmast-4-ene-3j6-dione₅ cholest^«5~ene- 1 ,3,1 1,18-tetrol, stigmasta-5,22- diene-3.7,16-triol, cholesterol and stigmasi-5-ene-3 7--dio!) and elevated levels of hydroxy fated

34 macyl glycerols and alkyl-aeylglycerols. The HMP dogs ("normal" memory dogs) had elevated levels of indoles-propanoic acid and leukotriene. C5 in comparison to the levels detected in the serum of the M P and LMP dogs. Elevated levels of a~Tocopherol (vitamin E) and cho!esteryl acetate were detected in the LMP dogs.

[0054} In the specification, there have been disclosed typical preferred embodiments of the invention. Althoug specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation. The scope of the invention is set forth in the claims. Obviously many modifications and variations of the invention are possible in light of the above teachings. It is therefore to be understood thai within the scope of the appended claims, the invention may be practiced, otherwise than as specifically described.

Claims

What is claimed is;

1 , A method for diagnosing cognitive impairment m an animal comprising:

a, obtaining a sample from the animal;

b. analyzing the sample for the presence of one or more metabolites associated with cogmti ve impairment;

c. comparing the amount of metabolites identified in the sample to a corresponding amount of the same metabolites present in a sample from one or more comparable control animals that do not suffer from cognitive impairment; and

d, using said comparison to diagnose cognitive impairment in the animal if the metabolites found in the animal^'s sample are greater or less than the amount present in the control sample.

2. The method of claim I wherein the sample is a serum sample.

3, The method of claim 1 wherein the diagnosis is based upon determining the amount of one metabolite associated with cognitive impairment,

4. The method of claim I wherein the diagnosis is based upon determining the amount of one or more metabolites associated with cognitive impairment.

5. The method of claim I wherei the diagnosis is based upon determining the amoun of one or more metabolites having an accurate mass of, or substantially equivalent to, 431.3845, 586.4545, 428.366, 429,3692, 859.9152, 783.5214, 600,47, 430.3815, 746,6425, 918.5546, 230.8621 , 476.3499, 300.266, 691 ,5464, 700,4985, 743.5836, 766,4501 , 801.0394, 1033.776, 658.4797, 690.5431_., 920.7392. 762.5803, 659.484, 620.5556, 603.5657, 189.0784, 212.1 1 15, 304.6394, 1085.962, 1056.984, 725.5073, 634.289, 755.4417, 549.623, 61 1.2828, 621.2718, 831.6701 , 264.1 04, 237.8986, 733.477, 193,883, 237.5638, 577.251 1 , 610.2803, 712.4157, 431.385, 678,278, 437.913, 612.553, 445.363, 287.77, 808.661 , 272.751, 291 ,931 , 312.23, 686,235, 606.257, 291.596, 607.261 , 658.48, 301.949, 653.267, 652.264.

6, The method of claim 1 wherein the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 431.3845, 586.4545, 428.366, 429.3692, 859.9152, 783.52 4, 600.47, 430.3815, 746,6425, 918.5546,

230.8621, 476.3499, 300.266, 691.5464, 700.4985, 743.5836, 766.4501 , 801.0.394,· 1033.776, 658.4797, 690,5431 , 920.7392. 762.5803, 659.484, and 620.5556.

The method of claim 1 wherein the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 603.5657, 189.0784, 212, 1 1 15, 304.6394, 1085.962, 230.8621 , 1056.984, 428.366, 725.5073, 586.4545, 634.289, 755.4417, 620.5556, 549.623, 600.47, 611.2828, 621.2718, 746.6425, 831 ,6701, 264, 1004, 7S3.5214, 237.8986, 691.5464, 429.3692, 733.477, 193,883, 237.5638, 577.251 , 10.2803, and 712.41 7,

The method of claim 1 wherein the diagnosis is based upon determining the amount of one or more metabolites having an. accurate mass of, or substantially equivalent to, 4 1 .385, 678.278, 437.913, 612.553, 445,363, 287.77, 808.661 , 272.751 , 291.931, 659,484, 312,23, 686.235. 606.257, 291 .596, 607.261, 658,48, 301 ,949, 653.267, and 652,264,

The method of claim I wherein the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 430.3 5, 428.366, 783.5214, 600,47, 746,6425, 918.5546, 230.8621 , 476.3499, and 300.266,

. The method of claim 1 wherein the animal is a human,

. The method of claim 1 wherein the animal is a companion animal.

.. The method of claim 1.1 wherein the companion animal is a dog.

, The method of claim 1 1 wherein the companion animal is a cat

. A method for determining if an animal is responding to treatment for cognitive impairment comprising;

a. obtaining a sample from the animal;

b. analyzing the sample for the presence of one or more metabolites associated with cognitive impairment;

c. comparing the amount of metabolites identified in the sample to a corresponding amount of the same metabolites present in previous sample from the same animal; and

d. using said comparison to determine an improvement in the animal if the metabolites found in the animal's sample are lower or higher than the amount present in the previous sample.

. The method of claim 14 wherein the sample is a serum, sample.

1 . The method of claim 14 wherein the diagnosis is based upon determining the amount of one -metabolite associated with cognitive impairment.

17. The method of claim 14 wherein the diagnosis is based upon determining the amount of one or more metabolites associated with cognitive impairment.

1 8. The method o claim 14 wherein the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 43.1.3845, 586.4545, 428.366, 429.3692, 859,9152, 783.5214, 600.47, 430.3815, 746.6425, 918.5546, 230.8621, 476.3499, 300.266, 691.5464, 700.4985, 743.5836, 766.4501 , 801.0394, 1033.776, 658.4797, 690.5431, 920.7392, 762.5803, 659.484, and 620.5556.

19. The method of claim 14 wherein the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 603,5657, 189.0784, 212, 1 1 15, 304.6394, 1085.962, 230.8621, 1056.984, 428.366, 725.5073, 586.4545, 634.289, 755.4417, 620.5556, 549.623, 600.47, 611.2828, 621.2718, 746.6425, 831.6701 , 264.1004, 783,5214, 237.8986, 691 .5464, 429.3692, 733.477, 193.883, 237.5638, 577.2511 , 610.2803, and 712.4157.

20. The method of claim 14 wherein the diagnosis is based upon determining the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 431.385, 678,278, 437,913, 612.553. 445.363, 287.77, 808.661 , 272.751 , 291.931 , 659,484, 312.23, 86.235, 606.257, 291.596, 607.261, 658.48, 301.949, 653.267, and 652.264.

21 . The method of claim 14 wherein the diagnosis is based upon deiemuning the amount of one or more metabolites having an accurate mass of, or substantially equivalent to, 430.3815, 428,366, 783.5214, 600,47, 746.6425, 918,5546, 230.8621 , 476.3499, and 300,266.

22. The method of claim 14 wherein the animal is a human.

23. The method of claim 14 wherein the animal is a companion animal.

24. The method of claim 23 wherein the companion animal is a dog.

25. The method of claim 23 wherein the companion animal is a cat.