WO2017149300A1 - Marqueurs pour troubles squelettiques - Google Patents

Marqueurs pour troubles squelettiques Download PDF

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Publication number
WO2017149300A1
WO2017149300A1 PCT/GB2017/050546 GB2017050546W WO2017149300A1 WO 2017149300 A1 WO2017149300 A1 WO 2017149300A1 GB 2017050546 W GB2017050546 W GB 2017050546W WO 2017149300 A1 WO2017149300 A1 WO 2017149300A1
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Prior art keywords
stage
skeletal
early
individual
markers
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PCT/GB2017/050546
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English (en)
Inventor
Paul J. THORNALLEY
Naila Rabbani
Richard Savage
Usman Ahmed
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The University Of Warwick
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Publication of WO2017149300A1 publication Critical patent/WO2017149300A1/fr

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/10Musculoskeletal or connective tissue disorders
    • G01N2800/101Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/10Musculoskeletal or connective tissue disorders
    • G01N2800/105Osteoarthritis, e.g. cartilage alteration, hypertrophy of bone
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/10Musculoskeletal or connective tissue disorders
    • G01N2800/108Osteoporosis

Definitions

  • the present invention relates to methods for determining the skeletal health of an individual by quantifying oxidised, nitrated and glycated free adducts, methods for determining whether an individual has an early-stage skeletal disorder by quantifying oxidised, nitrated and glycated free adducts, the use of said oxidised, nitrated and glycated free adducts as markers of skeletal health and early-stage skeletal disorder, as well as kits comprising reagents for quantifying said free adducts.
  • Osteoarthritis OA
  • RA rheumatoid arthritis
  • Severe life impairment may be prevented if decline in musculoskeletal health and development of OA and RA are identified and treated in the early-stages (Neogi, T. & Zhang, Y. Osteoarthritis Prevention. Current opinion in rheumatology 23, 185-191 (201 1); Isaacs, J. D. The changing face of rheumatoid arthritis: sustained remission for all? Nat Rev Immunol 10, 605-61 1 (2010)).
  • MRI Magnetic resonance imaging
  • MRI techniques require expensive instrumentation time and facilities, and they are contraindicated in certain populations who have implanted devices such as pacemakers or aneurysm coils.
  • eRA early-stage rheumatoid arthritis
  • RF rheumatoid factor
  • CCP anti-cyclic citrullinated peptide
  • a 4-class diagnostic algorithm was developed to detect and discriminate eOA, eRA and other inflammatory joint disease which may be self-resolving (non-RA) (WO 2014/016584).
  • This diagnostic algorithm combined measurement of hydroxyproline (Hyp), anti-CCP antibody and citrullinated protein (CP) in plasma with subject age and gender. Sensitivities and specificities were in the ranges 0.25 - 0.73 and 0.75 - 0.91 , respectively; with a random outcome value of 0.25.
  • Increased levels of CP are observed in both eOA and eRA with only autoimmunity in eRA, judged by anti-CCP antibody test positivity (Ahmed, U.
  • the present invention provides a method for determining the skeletal health of an individual comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • This aspect of the invention uses a 2-class diagnostic algorithm (eg. as illustrated in Figure 2(a)) to determine the skeletal health of a test individual, allowing reliable diagnosis of individuals having a skeletal disorder, including those having an early-stage skeletal disorder.
  • a 2-class diagnostic algorithm eg. as illustrated in Figure 2(a)
  • the present inventors have identified various subsets of markers that provide highly sensitive and specific determination of skeletal health.
  • the method of the invention allows the skeletal health of a test individual to be determined with a high level of sensitivity and specificity.
  • the invention further provides the use of at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK); dityrosine (DT); 3- nitrotyrosine (3-NT); N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp) for determining skeletal health.
  • markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO),
  • the invention also provides a method of treating a skeletal disorder in an individual, comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • the invention provides a method of determining whether an individual has an early- stage skeletal disorder comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ ⁇ - carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody;
  • MetSO methionine sulfoxide
  • DT dityrosine
  • 3-NT 3-nitrotyrosine
  • test individual has an early-stage skeletal disorder.
  • This aspect of the invention uses a 3-class diagnostic algorithm (as illustrated in Figure 2(b)) to reliably determine whether an individual has an early-stage skeletal disorder.
  • a 3-class diagnostic algorithm as illustrated in Figure 2(b) to reliably determine whether an individual has an early-stage skeletal disorder.
  • the present inventors have identified various subsets of markers that can be used in a diagnostic algorithm to distinguish different types of early-stage skeletal disorder with a high degree of sensitivity and specificity.
  • the method permits sensitive and specific distinction between different types of early-stage skeletal disorder, such as early-stage osteoarthritis, early-stage rheumatoid arthritis and inflammatory joint disease which may be self-resolving (see Figures 12 and 14).
  • There are currently no methods available in the art which allow the different types of early- stage skeletal disorder to be distinguished in a specific and sensitive manner. The method of the invention therefore provides a significant technical contribution to the art.
  • the invention also provides the use of at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl- lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody (anti-CCP antibody) for determining whether a test individual has an early-stage skeletal disorder.
  • markers selected from: the oxidised, nitrated, and glycated free
  • the invention also provides a method of treating an early-stage skeletal disorder in an individual, comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody;
  • the invention also provides a method of determining whether an individual has an early-stage skeletal disorder comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT),
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody;
  • This method provides a highly sensitive way of detecting early-stage skeletal disorder using a combination of 2 diagnostic algorithms.
  • the first algorithm is used to determine the skeletal health of an individual, thereby identifying individuals having a skeletal disorder.
  • the second algorithm is used to determine whether the skeletal disorder is an early-stage skeletal disorder, and may be used to distinguish between specific types of early-stage skeletal disorder such as early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self- resolving.
  • eOA early-stage osteoarthritis
  • eRA early-stage rheumatoid arthritis
  • This method provides the most sensitive and specific approach to determining whether an individual has an early-stage skeletal disorder (as shown in Figures 12 and 14).
  • the invention also provides a method of treating an early-stage skeletal disorder in an individual, comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp); and classifying the skeletal health based on the amount of each marker quantified in
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody; and classifying the skeletal health based on the amount
  • the invention provides a method for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis, comprising:
  • the method may comprise the step of classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm, wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • the invention further provides the use of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3- NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), and N £ -fructosyl-lysine (FL) as markers for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis.
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine (3- NT)
  • CMA ⁇ ⁇ -
  • the invention provides a useful way of monitoring the skeletal health of the test individual over time to determine whether the skeletal health of the test individual has improved, worsened, or remained stable.
  • the invention provides a method for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis, comprising:
  • the invention further provides the use of the combination of the oxidised free adducts: methionine sulfoxide (MetSO), and dityrosine (DT), and the glycated free adduct pentosidine as markers for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis.
  • MethodSO methionine sulfoxide
  • DT dityrosine
  • the method may comprise the step of classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm, wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • the invention provides a kit comprising reagents for quantifying markers of skeletal health, wherein said markers comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3- nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3-de
  • markers comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT
  • the invention also provides a kit comprising reagents for quantifying markers of early-stage skeletal disorder, wherein said markers comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl- lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane, and/or related stable isotype substituted compounds (isotopomers); and anti-cyclic citrullinated peptide antibody.
  • markers comprise at
  • the invention also provides a kit comprising reagents for quantification of markers for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis, wherein said markers comprise: the combination of the oxidised, nitrated and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), and N £ -fructosyl-lysine (FL); and/or related stable isotype substituted compounds (isotopomers).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine (3-NT)
  • the invention also provides a kit comprising reagents for quantification of markers for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis, wherein said markers comprise the combination of the oxidised free adducts: methionine sulfoxide (MetSO), and dityrosine (DT), and the glycated free adduct pentosidine; and/or related stable isotype substituted compounds (isotopomers).
  • MetSO methionine sulfoxide
  • DT dityrosine
  • the invention also provides a computational model based on a diagnostic algorithm adapted to classify the skeletal health based on the amount of a plurality of markers quantified in a test sample with a diagnostic algorithm, wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • Said markers may comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3- NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3- NT
  • Said markers may comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody.
  • MetSO methionine sulfoxide
  • DT dityrosine
  • 3-NT 3-nitrotyrosine
  • the invention also extends to software adapted to produce a computational model as aforementioned.
  • the invention also extends to a processor adapted to produce a computational model as aforementioned.
  • the invention also provides a method
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody;
  • the present inventors have conducted extensive experimentation to identify markers of skeletal health.
  • the present inventors have identified various sets of oxidised, nitrated, and glycated free adducts and adduct residues of proteins that can be used to determine the skeletal health of an individual, and to determine whether an individual has an early-stage skeletal disorder, or to distinguish between advanced and early-stage skeletal disorders with a high degree of specificity and sensitivity.
  • the present invention provides a method for determining the skeletal health of an individual comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • determining the skeletal health of an individual means determining whether an individual has a skeletal disorder, or does not have a skeletal disorder.
  • the terms "individual”, “subject” and “patient” are used interchangeably herein to refer to a mammalian subject whose skeletal health requires investigation.
  • the mammal can be a human, or an animal including, but not limited to an equine, porcine, canine, feline, ungulate, and primate animal, or any other load-bearing animal that is known to be susceptible to, or suffer from, a skeletal health disorder.
  • the individual is a human.
  • the methods and uses of the invention described herein are useful for both medical and veterinary uses.
  • the individual may not have been previously diagnosed as having a skeletal disorder.
  • the individual may also be one who has been previously diagnosed as having the disorder (ie. for advanced stage skeletal disorders).
  • the individual may be one who does exhibit disease risk factors, or one who is asymptomatic for the disease (ie. for early-stage skeletal disorders).
  • the individual may also be one who is suffering from or a risk of developing a skeletal disorder.
  • skeletal disorder means any joint or bone disorder, or a condition that gives rise to a lack of skeletal health or integrity.
  • joint or bone disorders include arthritic conditions, such as non-inflammatory arthritic conditions ("non-inflammatory arthritis") and inflammatory arthritic conditions ("inflammatory arthritis”).
  • Non-inflammatory arthritic conditions include without limitation osteoarthritis (OA), such as early-stage OA (eOA) or advanced-stage OA (aOA).
  • Inflammatory arthritic conditions include without limitation rheumatoid arthritis, such as early-stage RA (eRA) or advanced-stage RA (aRA).
  • Skeletal disorders may also include inflammatory joint disorders which may be self-resolving (non-RA).
  • the method of the invention is used to diagnose the test individual as having a skeletal disorder selected from at least one of: early-stage osteoarthritis (eOA), advanced-stage osteoarthritis (aOA), early-stage rheumatoid arthritis (eRA), advanced-stage rheumatoid arthritis (aRA), or other inflammatory joint disease that may be self-resolving.
  • the method of the invention is used to diagnose the test individual as having a skeletal disorder selected from at least one of: early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving.
  • the term "early-stage skeletal disorder” refers to a skeletal disorder that is in the early stages of the disease condition.
  • the individual may experience no symptoms of the skeletal disorder.
  • the individual may experience temporary symptoms.
  • the individual may experience mild symptoms.
  • the early-stage skeletal disorder may be early-stage osteoarthritis.
  • Individuals having eOA may experience no join pain. Individuals may experience pain after prolonged use of a joint. The pain may be mild. The individuals may experience stiffness after prolonged non-use of the joint. Radiographs of the joints in the individual may appear normal. The individual may have minimal cartilage breakdown. The individual may be classified as grade I or grade II on the Outerbridge scale.
  • the joints commonly affected in OA are of the spine, fingers, thumbs, hips, knees and toes
  • the early-stage skeletal disorder may be early-stage rheumatoid arthritis.
  • Individuals having eRA may experience temporary joint pain and joint swelling during or after use. Radiographs of the joints in the individual may appear normal.
  • the individual may have minimal cartilage breakdown.
  • the appearance of the joint may be normal.
  • the joints commonly affected in RA are of the hand, wrist, shoulder, elbow, knee, ankle and feet. Accordingly, the methods of the invention as described herein, permit diagnosis of individuals that may not have been previously diagnosed as having a skeletal disorder.
  • the individual may not exhibit disease risk factors or may be asymptomatic for the disease.
  • the term "advanced-stage skeletal disorder" refers to a skeletal disorder that is in the later stages of the disease condition.
  • the individual may experience persistent symptoms of the skeletal disorder.
  • the individual may experience severe symptoms.
  • the advanced-stage skeletal disorder may be advanced-stage osteoarthritis.
  • Individuals having aOA may experience frequent join pain during or after use (eg. when walking, running, bending or kneeling).
  • Individuals may experience join swelling after prolonged use (eg. when walking, running, bending or kneeling).
  • the pain may be severe.
  • Individuals may experience joint stiffness that worsens after prolonged non-use of the joint (eg. after sitting for a prolonged period or when waking up in the morning).
  • Radiographs of the joints in the individual may indicate breakdown and change in surface morphology of bone in joints.
  • the individual may be classified as grade III or grade IV on the Outerbridge scale.
  • the advanced-stage skeletal disorder may be advanced-stage rheumatoid arthritis.
  • Individuals having aRA may experience persistent joint pain.
  • Individuals may experience joint swelling.
  • Individuals may experience a limited range of motion in the joint.
  • Individuals may experience stiffness in the joint (particularly in the morning).
  • Individuals may experience weakness and malaise.
  • Radiographs of the joints in the individual may indicate breakdown and change in surface morphology of bone in joints.
  • the joints in the individual may be deformed.
  • the individual may have muscle atrophy.
  • an "inflammatory joint disease that is self- resolving” refers to an inflammatory skeletal disorder that is mild in severity and often short in duration, resolving without major treatment.
  • etiology are: reactive arthritis (joint pain and swelling triggered by an infection in another part of your body), pseudogout (caused by deposits of crystals of calcium pyrophosphate in and around the joints), and other unclassified conditions.
  • the method of the invention is used to diagnose the test individual as having no skeletal disorder
  • body fluid sample includes a sample obtained from eye fluid, urine, whole blood, blood serum, blood plasma, lymphatic fluid, saliva, synovial fluid, seminal fluid, cerebrospinal fluid, sebaceous secretions, or sputum.
  • said sample may be pre-treated for analysis, typically, by using conventional techniques as described herein and known by those skilled in the art.
  • the body fluid sample is urine. In one embodiment, the body fluid sample is sputum. In one embodiment, the body fluid sample is selected from blood serum, blood plasma and synovial fluid. In one embodiment, the body fluid sample is a synovial fluid sample. In one embodiment, the body fluid sample is a blood serum sample. In one embodiment, the body fluid sample is a blood plasma sample.
  • a key advantage to using blood plasma or blood serum in the methods of the invention is that these samples are readily available and can be obtained using minimally invasive techniques. This is particularly advantageous when attempting to diagnose early-stage skeletal disorder.
  • the phrase "quantifying markers of skeletal health in a body fluid sample” means determining the amount of the markers that are present in the body fluid sample of a test individual.
  • the amount of the markers that are present in the body fluid sample this means quantifying the marker by determining, for example, the relative or absolute amount of the marker.
  • the assay methods do not necessarily require measurement of absolute values of marker, unless it is desired, because relative values are sufficient for many applications of the invention.
  • the "amount" can be the (absolute) total amount of the marker that is detected in a sample, or it can be a "relative" amount, e.g., the difference between the marker detected in a sample and e.g. another constituent of the sample.
  • the amount of the marker may be expressed by its concentration in a sample, or by the concentration of a reagent that detects the marker.
  • the methods of the present invention may determine the amount of each marker.
  • the methods of the invention may determine the cumulative amount of all the markers.
  • the amount of the markers can be combined with each other in a formula to form an index value.
  • Oxidised, nitrated and glycated free adducts and adduct residues are markers of impaired skeletal health. Oxidative and nitration damage to proteins (such as those present in joint tissue) in arthritis arises as a consequence of increased reactive oxygen species (ROS) in the phagocytic respiratory burst of phagocytes in cell-mediated inflammatory response, and during mitochondrial dysfunction (Wright, H. L, Moots, R.
  • ROS reactive oxygen species
  • oxidative stress through: (i) increased advanced glycation endproducts (AGEs) formed by oxidative processes - glycoxidation adducts such as CML, (ii) decreased metabolism of dicarbonyl precursors glyoxal, methylglyoxal and 3-deoxyglucosone (which increase formation of dicarbonyl-derived AGEs, CMA, G-H1 , MG-H1 , and 3DG-H), and (iii) and increased pentosephosphate pathway activity countering oxidative stress (increasing formation of trace pentose dicarbonyl precursor of pentosidine) (Thornalley, P. J. & Rabbani, N.
  • AGEs advanced glycation endproducts
  • proteins having oxidised, nitrated and glycated adduct residues.
  • proteolysis When these proteins undergo proteolysis, they release oxidised, nitrated and glycated free adducts, which transit into plasma for clearance in the kidney and eventual excretion in urine.
  • the adduct residues (attached to protein) and free adducts (released from protein) thus provide markers that relate to directly impaired skeletal health.
  • the oxidised, nitrated and glycated free adducts and adduct residues of proteins therefore provide suitable markers for determining skeletal health of individuals.
  • oxidised, nitrated, and glycated free adducts refers to the proteolytic digestion products that have been released into the body fluid of the test individual following proteolysis of oxidised, nitrated and glycated proteins.
  • the pre-analytic processing steps required to obtain a sample of oxidised, nitrated and glycated free adducts are simple and rapid. Oxidised, nitrated and glycated free adducts therefore provide ideal markers for use in the methods of the invention.
  • oxidised, nitrated, and glycated free adducts can instead be quantified in the method of determining the skeletal health of an individual.
  • the phrase “oxidised, nitrated, and glycated adduct residues” refers to the oxidised, nitrated and glycated adduct residues of proteins that are present in the body fluid of the test individual.
  • oxidised, nitrated, and glycated adduct residues may also be used in all methods, uses and kits of the invention.
  • the reference to "oxidised, nitrated, and glycated free adducts" may be replaced with a reference to "oxidised, nitrated, and glycated adduct residues".
  • Example 2 the present inventors have demonstrated that by quantifying specific sub-sets of oxidised, nitrated and glycated free adducts (or adduct residues), it is possible to determine the skeletal health of an individual with a high degree of specificity and sensitivity, which advantageously allows early diagnosis of skeletal disorder.
  • Hydroxyproline is a known marker of skeletal health (Ahmed, U. et al. Biomarkers of early stage osteoarthritis, rheumatoid arthritis and musculoskeletal health. Sci. Rep. 5, 9259 (9251 -9257) (2015)).
  • two sub-sets of markers have been identified as providing excellent results. Good results can, however, still be obtained when using fewer markers.
  • the markers of skeletal health that are quantified in the method for determining skeletal health may comprise at least 6 (eg. at least 7, at least 8, at least 9, at least 10, at least 1 1 , or all 12) markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal- derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp).
  • MetSO methionine
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine and glucosepane; and hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine and ⁇ ⁇ -carboxymethylarginine (CMA); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: N-formylkynurenine (NFK), dityrosine (DT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and hydroxyproline
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine and N-formylkynurenine (NFK); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts): ⁇ ⁇ -carboxymethylarginine (CMA), dityrosine (DT), N £ -(1 -carboxyethyl)lysine (CEL), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and hydroxypro
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA) and N-formylkynurenine (NFK); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), ⁇ ⁇ - (l -carboxyethyl)lysine (CEL), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) and N £ -(1 -carboxyethyl)lysine (CEL); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and hydroxyproline
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), and methylglyoxal-derived hydroimidazolone (MG-H1 ), and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), and N e -fructosyl-lysine (FL); and hydroxypro
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), and dityrosine (DT); and optionally further comprise one or more markers selected from: the glycated free adduct glyoxal-derived hydroimidazolone (G-H1) and N e -fructosyl-lysine (FL); and hydroxyproline (Hyp).
  • MetSO
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), and glyoxal-derived hydroimidazolone (G-H1), and optionally further comprise N e -fructosyl-lysine (FL) and/or hydroxyproline (Hyp).
  • MetalSO methionine sulfoxide
  • 3DG-H 3-
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), and N e -fructosyl-lysine (FL), and optionally further comprise glyoxal-derived hydroimidazolone (G-H1) and/or hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • 3DG-H 3-de
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), and N e -fructosyl-lysine (FL), and optionally further comprise hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxygluco
  • all combinations of markers described herein may additionally comprise the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the order of utility of the skeletal markers for diagnostic performance of the algorithm is as follows:
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), and glucosepane (GSP); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal- derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), and ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL);and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), N £ -(1 -carboxyethyl)lysine (CEL), and methylglyoxal-derived hydroimidazolone (MG-H1);and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1 ), and N e -fructosyl-lysine (FL); and hydroxy
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), and dityrosine (DT); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: glyoxal-derived hydroimidazolone (G-H1), and N e -fructosyl-lysine (FL); and hydroxy
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), and N e -fructosyl-lysine (FL); and optionally further comprise glyoxal-derived hydroimidazolone (G-H1) and/or hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • 3DG-H 3-
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), and glyoxal-derived hydroimidazolone (G-H1); and optionally further comprise N e -fructosyl-lysine (FL) and/or hydroxyproline (Hyp).
  • MetalSO methionine sulfoxide
  • 3DG-H
  • the markers of skeletal health that are quantified in the method for determining skeletal health comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), and N e -fructosyl-lysine (FL); and optionally further comprise hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglu
  • all combinations of markers described herein may additionally comprise the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the above combination of markers may also be used as markers of early-stage skeletal disorder in the method described herein. Measurement of the markers can be performed by any method that provides satisfactory analytical specificity, sensitivity and precision. The invention thus encompasses the use of those methods known to a person skilled in the art to measure the amount of the free adduct or adduct residues in a body fluid sample.
  • the free adducts or adduct residues may be quantified by liquid chromatography- tandem mass spectrometry, such as stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC- MS/MS).
  • liquid chromatography- tandem mass spectrometry such as stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC- MS/MS).
  • LC- MS/MS stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry
  • the method may further comprise an initial step of isolating the oxidised, nitrated, and glycated free adducts from the body fluid sample by ultrafiltration.
  • the ultrafiltrate sample (containing the free adducts) is collected and used in the quantification step of the method.
  • the oxidised, nitrated and glycated free adducts are collected by microspin ultrafiltration. A molecular weight cut-off of at least about 10kDa may be used in the ultrafiltration step.
  • the molecular weight cut-off may be at least about 5kDa (such as at least about 6kDa, 7kDa, 8kDa, 9kDa, 10kDa, 1 1 kDa, 12kDa, 13kDa, 14kDa or 15kDa).
  • the ultrafiltration step may be performed at a temperature of between about 2°C and 10°C, such as at about 4°C.
  • the method may further comprise an initial step of hydrolysing the adduct residues to release amino acids for quantification.
  • the hydrolysis is performed enzymatically. Protein hydrolysis by enzymatic digestion is advantageous because it avoids the severe conditions of acid hydrolysis which may compromise the analyte content of the sample during pre-analytic processing.
  • enzymatic digestion may involve treatment with pepsin, followed by treatment with pronase E, prolidase and aminopeptidase.
  • collagenase may be used, particularly, but not exclusively, where the protein to be assayed is present in the extracellular matrix.
  • Automated exhaustive enzymatic hydrolysis may be used, thereby avoiding harsh, pre-analytic processing.
  • the proteins prior to hydrolysis, the proteins may be first washed by ultrafiltration to remove free amino acids, and retained protein is collected for hydrolysis.
  • the retained protein may be delipidated prior to hydrolysis.
  • the oxidised, nitrated and glycated adduct residues may be normalised to their amino acid residue precursors and given as mmol/mol amino acid modified.
  • Hydroxyproline may be quantified using routine immunoassays known to the skilled person.
  • the hydroxyproline (Hyp) marker may be quantified using liquid chromatography-tandem mass spectrometry.
  • the hydroxyproline (Hyp) marker is quantified using stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC- MS/MS), as described above.
  • the hydroxyproline (Hyp) quantified in the body fluid is free (dialyzable) hydroxyproline (Hyp).
  • the body fluid sample used for quantification of hydroxyproline (Hyp) may be the same sample used to quantify the oxidised, nitrated and glycated free adducts or adduct residues, or it may be a different body fluid sample obtained from the test individual.
  • the body fluid sample used to quantify Hyp may be a urine sample.
  • the sample may be further assayed for creatinine, and the amount of Hyp in the test sample is normalised, having regard to the amount of creatinine present in said sample.
  • the method may further comprise an initial step of isolating the hydroxyproline (Hyp) marker from the body fluid sample by ultrafiltration prior to quantification, as described above.
  • the ultrafiltrate sample (containing the hydroxyproline) is collected and used in the quantification step of the method.
  • the method of the invention for determining the skeletal health of an individual may further comprise the step of quantifying anti-cyclic citrullinated peptide antibody (anti-CCP antibody).
  • anti-CCP antibody anti-cyclic citrullinated peptide antibody
  • the body fluid sample used for quantification of anti-CCP antibody may be the same sample used to quantify the other markers of skeletal health, or it may be a different body fluid sample obtained from the test individual.
  • the method of determining the skeletal health of an individual further comprises quantifying rheumatoid factor (RF) in a body fluid sample obtained from the test individual.
  • RF rheumatoid factor
  • the method of determining the skeletal health of an individual further comprises quantifying citrullinated proteins in a body fluid sample obtained from the test individual. Further discussion of suitable methods for detecting citrullinated proteins may be found in WO 2014/016584.
  • the method of the invention for determining the skeletal health of an individual may further comprise including the age and/or gender of the test individual as further markers.
  • Quantification of gender may for example comprise assigning a value of 1 if the test individual is female, and a value of 0 if the test individual is male.
  • the phrase "comparing the amount of each marker quantified in the test sample to corresponding reference values for each marker in a diagnostic algorithm” refers to the comparative process by which the amount of a marker quantified in the test sample is compared to a reference value for the same marker using a diagnostic algorithm.
  • the comparative process may be part of a classification by a diagnostic algorithm.
  • the comparative process may occur at an abstract level, e.g. in n-dimensional feature space or in a higher dimensional space.
  • the term "reference value” refers to a value obtained from a population of individual(s) whose disease state is known.
  • the reference value may be in n-dimensional feature space and may be defined by a maximum-margin hyperplane.
  • a reference value can be determined for any particular population, subpopulation, or group of individuals according to standard methods well known to those of skill in the art.
  • the phrase "classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm” refers to the statistical or machine learning classification process by which the amount of a marker quantified in the test sample is used to determine a category of skeletal health with a diagnostic algorithm, typically a statistical or machine learning classification algorithm.
  • Classification by a diagnostic algorithm may include scoring likelihood of a panel of marker values belonging to each possible category, and determining the highest-scoring category. Classification by a diagnostic algorithm may include comparing a panel of marker values to previous observations by means of a distance function. Examples of diagnostic algorithms suitable for classification include random forests, support vector machines, logistic regression (e.g. multiclass or multinomial logistic regression, and/or algorithms adapted for sparse logistic regression). A wide variety of other diagnostic algorithms that are suitable for classification may be used, as known to a person skilled in the art.
  • training the diagnostic algorithm may refer to supervised learning of a diagnostic algorithm on the basis of values for each marker obtained from a population of individuals having known skeletal health.
  • the term "population of individuals” means one or more individuals. In one embodiment, the population of individuals consists of one individual. In one embodiment, the population of individuals comprises multiple individuals. As used herein, the term “multiple” means at least 2 (such as at least 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30) individuals. In one embodiment, the population of individuals comprises at least 10 individuals.
  • the reference value is the amount of a marker in a sample or samples derived from one individual.
  • the reference value may be derived by pooling data obtained from multiple individuals, and calculating an average (for example, mean or median) amount for a marker.
  • the reference value may reflect the average amount of a marker in multiple individuals. Said amounts 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.
  • the reference value may be derived from the same sample as the sample that is being tested, thus allowing for an appropriate comparison between the two.
  • the sample is derived from urine
  • the reference value is also derived from urine.
  • the sample is a blood sample (e.g. a plasma or a serum sample)
  • the reference value will also be a blood sample (e.g. a plasma sample or a serum sample, as appropriate).
  • the way in which the amounts are expressed is matched between the sample and the reference value.
  • an absolute amount can be compared with an absolute amount
  • a relative amount can be compared with a relative amount.
  • the way in which the amounts are expressed for classification with the diagnostic algorithm is matched to the way in which the amounts are expressed for training the diagnostic algorithm.
  • the method may comprise comparing the amount of each marker to its corresponding reference value.
  • the method may comprise comparing the cumulative amount to a corresponding reference value.
  • the index value can be compared to a corresponding reference index value derived in the same manner.
  • the reference values may be obtained either within (ie. constituting a step of) or external to the (ie. not constituting a step of) methods of the invention.
  • the methods of the invention may comprise a step of establishing a reference value for the quantity of the markers.
  • the reference values are obtained externally to the method of the invention and accessed during the comparison step of the invention.
  • the training of a diagnostic algorithm may be obtained either within (ie. constituting a step of) or external to (ie. not constituting a step of) the methods of the invention.
  • the methods of the invention may comprise a step of training of a diagnostic algorithm.
  • the diagnostic algorithm is trained externally to the method of the invention and accessed during the classification step of the invention.
  • the reference value may be determined by quantifying the amount of a marker in a sample obtained from a population of healthy individual(s).
  • the diagnostic algorithm may be trained by quantifying the amount of a marker in a sample obtained from a population of healthy individual(s).
  • the term "healthy individual” refers to an individual or group of individuals who are in a healthy state, e.g.
  • the healthy individual(s) is not on medication affecting the disease and has not been diagnosed with any other disease.
  • the one or more healthy individuals may have a similar sex, age and body mass index (BMI) as compared with the test individual.
  • the reference value may be determined by quantifying the amount of a marker in a sample obtained from a population of individual(s) suffering from the disease.
  • the diagnostic algorithm may be trained by quantifying the amount of a marker in a sample obtained from a population of individual(s) suffering from the disease. More preferably such individual(s) may have similar sex, age and body mass index (BMI) as compared with the test individual.
  • the reference value may be obtained from a population of individuals suffering from early-stage skeletal disorders (such as early-stage osteoarthritis or early-stage rheumatoid arthritis), or advanced-stage skeletal disorders (such as advanced-stage osteoarthritis or advanced -stage rheumatoid arthritis).
  • the diagnostic algorithm may be trained by quantifying the amount of a marker in a sample obtained from a population of individuals suffering from early-stage skeletal disorders (such as early-stage osteoarthritis or early-stage rheumatoid arthritis), or advanced-stage skeletal disorders (such as advanced-stage osteoarthritis or advanced -stage rheumatoid arthritis).
  • the markers quantities characteristic of early-stage skeletal disorder may, in fact, be determined only by a retrospective analysis of samples obtained from individuals who ultimately manifest clinical symptoms of the skeletal disorder.
  • the known skeletal health may be determined only by a retrospective analysis of samples obtained from individuals who ultimately manifest clinical symptoms of the skeletal disorder.
  • the characteristic marker profile of an early-stage skeletal disorder is determined, the profile of markers from a biological sample obtained from an individual may be compared to this reference profile to determine whether the test subject is also at that particular stage of the skeletal disorder.
  • the diagnostic algorithm is trained to classify early-stage skeletal disorder, the profile of markers from a biological sample obtained from an individual may be classified by the diagnostic algorithm to determine whether the test subject is also at that particular stage of the skeletal disorder.
  • the population of individuals used to obtain reference values for the diagnostic algorithm, and/or the population of individuals used to train the diagnostic algorithm may comprise: at least one healthy individual having no skeletal disorder, and/or at least one individual having a skeletal disorder selected from at least one of: early-stage osteoarthritis (eOA), advanced-stage osteoarthritis (aOA), early-stage rheumatoid arthritis (eRA), advanced-stage rheumatoid arthritis (aRA), or other inflammatory joint disease that may be self-resolving.
  • the population of individuals may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder, and/or multiple (eg.
  • individuals having a skeletal disorder selected from at least one of: early-stage osteoarthritis (eOA), advanced-stage osteoarthritis (aOA), early-stage rheumatoid arthritis (eRA), advanced-stage rheumatoid arthritis (aRA), or other inflammatory joint disease that may be self-resolving.
  • eOA early-stage osteoarthritis
  • aOA advanced-stage osteoarthritis
  • eRA early-stage rheumatoid arthritis
  • aRA advanced-stage rheumatoid arthritis
  • the population of individuals used to obtain reference values for the diagnostic algorithm, and/or the population of individuals used to train the diagnostic algorithm may comprise: at least one healthy individual having no skeletal disorder, at least one individual having early-stage osteoarthritis (eOA), at least one individual having advanced-stage osteoarthritis (aOA), at least one individual having early-stage rheumatoid arthritis (eRA), at least one individual having advanced-stage rheumatoid arthritis (aRA), and/or at least one individual having another inflammatory joint disease that may be self-resolving.
  • the population of individuals may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder, multiple (eg.
  • eOA early-stage osteoarthritis
  • aOA advanced-stage osteoarthritis
  • eRA early-stage rheumatoid arthritis
  • aRA advanced-stage rheumatoid arthritis
  • aRA advanced-stage rheumatoid arthritis
  • the population of individuals used to obtain reference values for the diagnostic algorithm, and/or the population of individuals used to train the diagnostic algorithm may comprise: at least one healthy individual having no skeletal disorder, at least one individual having early-stage osteoarthritis (eOA), at least one individual having early-stage rheumatoid arthritis (eRA), and/or at least one individual having another inflammatory joint disease that may be self-resolving.
  • the population of individuals may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder, multiple (eg. at least 10) individuals having early-stage osteoarthritis (eOA), multiple (eg. at least 10) individuals having early-stage rheumatoid arthritis (eRA), and/or multiple (eg. at least 10) individuals having another inflammatory joint disease that may be self-resolving.
  • samples obtained from individuals that have a skeletal disorder include those having early and advanced stage skeletal disorders
  • individuals having no skeletal disorder have different marker profiles (ie. the abundance of the markers quantified varies between the samples).
  • These differences in marker abundance between individuals having a skeletal disorder and those not having a skeletal disorder provides a way to classify individuals as having a skeletal disorder or not having a skeletal disorder by determining which marker profile they display.
  • the method permits classification of the individual as belonging to or not belonging to the reference population (ie. by determining whether the amounts of marker quantified in the individual are statistically similar to the reference population or statistically deviate from the reference population).
  • classification of the individual's marker profile ie. the overall pattern of change observed for the markers quantified
  • classification of the individual's marker profile is predictive that the patient falls (or does not fall) within the reference population.
  • an individual may be diagnosed as having a skeletal disorder when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having a skeletal disorder. In one embodiment, an individual may be diagnosed as having no skeletal disorder when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having no skeletal disorder.
  • the term “statistically similar” means that the amounts of marker quantified for the test individual are similar to those quantified for the reference population to a statistically significant level.
  • the term “statistically significant” means that the alteration is greater than what might be expected to happen by chance alone. Statistical significance can be determined by any method known in the art.
  • an individual may be diagnosed as having a skeletal disorder when the amount of markers quantified statistically deviates from the amount determined for the corresponding values obtained from a population of individuals having no skeletal disorder. In one embodiment, an individual may be diagnosed as having no skeletal disorder when the amount of markers quantified statistically deviates from the amount determined for the corresponding values obtained from a population of individuals having a skeletal disorder.
  • the term "statistically deviates” means that the amounts of marker quantified for the test individual differs from those quantified for the reference population to a statistically significant level.
  • the deviation in marker abundance may be an increase or decrease.
  • comparing the amount of the marker relative to the reference value and determining an increase indicates that the individual has a skeletal disorder.
  • the increase can be, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140% or at least 150% of the reference value.
  • the increase in the amount of the markers may be statistically significant.
  • comparing the amount of the marker relative to the reference value and determining a decrease indicates that the individual has a skeletal disorder.
  • the decrease can be, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the reference value.
  • the decrease in the amount of the marker may be statistically significant.
  • comparing the amount of the marker relative to the reference value and determining an increase indicates that the individual does not have a skeletal disorder.
  • the increase can be, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 140% or at least 150% of the reference value.
  • the increase in the amount of the marker may be statistically significant.
  • comparing the amount of the marker relative to the reference value and determining a decrease indicates that the individual does not have a skeletal disorder.
  • the decrease can be, for example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the reference value.
  • the decrease in the amount of the markers may be statistically significant.
  • Classification of the individual in the methods of the invention is performed using a diagnostic algorithm.
  • the diagnostic algorithm used in the method of the invention is a classification algorithm.
  • the classification algorithm is an ensemble algorithm comprising different types of classification algorithms.
  • the classification algorithm comprises a decision tree based algorithm.
  • the classification algorithm comprises a support vector machine algorithm. Other types of algorithms, such as regression algorithms and neural networks, may also be used.
  • the classification algorithm comprises a random forest algorithm.
  • the diagnostic algorithm used in the method of the invention for determining the skeletal health of an individual may be a 2-class algorithm (e.g. see Figure 2(a)).
  • Classification of the individual by the diagnostic algorithm does not require perfect classification. Classification may be characterized by its "sensitivity.”
  • the "sensitivity” of classification relates to the percentage of individuals who were correctly identified as having a skeletal disorder, or in the case of determining whether an individual has an early-stage skeletal disorder, the percentage of individuals correctly identified as having a particular early-stage skeletal disorder.
  • Sensitivity is defined in the art as the number of true positives divided by the sum of true positives and false negatives.
  • the sensitivity of the methods of the invention may be at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, or at least about 65%.
  • the “specificity” of the methods of the invention is defined as the percentage of patients who were correctly identified as not having a skeletal disorder, or in the in the case of determining whether an individual has an early-stage skeletal disorder, the percentage of individuals correctly identified as not having a particular early-stage skeletal disorder. "Specificity” relates to the number of true negatives divided by the sum of true negatives and false positives. The specificity of the methods of the invention may be at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, or at least about 65%.
  • the invention further provides the use of at least 6 (eg. at least 7, at least 8, at least 9, at least 10, at least 1 1 , or all 12) markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp) for determining skeletal health.
  • markers selected from: the oxidised, nitrated,
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and hydroxyproline (Hyp) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and hydroxyproline (Hyp) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitro
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine and ⁇ ⁇ -carboxymethylarginine (CMA) as markers for determining skeletal health.
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine and N-formylkynurenine (NFK) as markers for determining skeletal health.
  • MetSO methionine sulfoxide
  • 3DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3-nitrotyrosine
  • pentosidine pentosidine
  • NFK N-formylkynurenine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA) and N-formylkynurenine (NFK) as markers for determining skeletal health.
  • MeSO methionine sulfoxide
  • 3DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • pentosidine ⁇ ⁇ -carboxymethylarginine (CMA) and N-formylkynurenine (NFK)
  • CMA ⁇ ⁇ -carboxymethylarginine
  • NFK N-formylkynurenine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) and ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL) as markers for determining skeletal health.
  • MeSO methionine sulfoxide
  • 3DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • pentosidine ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) and ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL) as markers for
  • the invention provides the use of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG- H), 3-nitrotyrosine (3-NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), ⁇ ⁇ - (l -carboxyethyl)lysine (CEL) and methylglyoxal-derived hydroimidazolone (MG-H1) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG- H 3-deoxyglucosone-derived hydroimidazolone
  • 3-NT 3-nitrotyrosine
  • pentosidine ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), ⁇ ⁇ - (
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts of methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1) and dityrosine (DT) as markers for determining skeletal health.
  • MethodSO methionine sulfoxide
  • 3DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • pentosidine ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), N
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT) and glyoxal-derived hydroimidazolone (G-H1) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • pentosidine ⁇ ⁇ -carboxymethylarg
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), and N £ -fructosyl-lysine (FL) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • the combination of markers described herein may additionally include the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), and glucosepane (GSP) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • CMA ⁇ ⁇ -carboxymethylarginine
  • NFK N-formylkynurenine
  • GSP glucosepane
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3-NT), ⁇ ⁇ - carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), and ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL) as markers for determining skeletal health.
  • (MetSO) 3-deoxyglucosone-derived hydroimidazolone
  • 3-NT 3-nitrotyrosine
  • CMA ⁇ ⁇ - carboxymethylarginine
  • NFK N-formylkynurenine
  • GSP glucosepane
  • CEL ⁇ ⁇ -(1 - carboxyethyl)lysine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), and methylglyoxal-derived hydroimidazolone (MG-H1) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • CMA ⁇ ⁇ -carboxymethylarginine
  • NFK N-formylkynurenine
  • GSP glucosepane
  • CEL
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), and dityrosine (DT) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • CMA ⁇ ⁇ -carboxymethylarginine
  • NFK N-formylkynurenine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), and glyoxal-derived hydroimidazolone (G-H1) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), and ⁇ ⁇ - fructosyl-lysine (FL) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • ⁇ ⁇ -carboxymethylarginine C
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), glyoxal- derived hydroimidazolone (G-H1) and N £ -fructosyl-lysine (FL) as markers for determining skeletal health.
  • Methionine sulfoxide MetalSO
  • 3-DG-H 3-deoxyglucosone-derived hydroimidazolone
  • the combination of markers described herein may additionally include the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • AASA oxidised free adducts or adduct residues
  • GSA GSA
  • the invention also provides a method of treating a skeletal disorder in an individual, comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • the "skeletal disorder” may be early-stage osteoarthritis (eOA), advanced-stage osteoarthritis (aOA), early-stage rheumatoid arthritis (eRA), advanced-stage rheumatoid arthritis (aRA), or other inflammatory joint disease that may be self-resolving.
  • the "skeletal disorder” may be early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving.
  • the administration of a treatment to the individual improves the skeletal health of the individual.
  • the phrase "improve the skeletal health of the individual” refers to the improvement in skeletal health of the individual identified as having a skeletal disorder (such as an early- stage or advanced-stage skeletal disorder) by administrating a treatment for that skeletal disorder.
  • the progression of the skeletal disorder from an early-stage disorder to an advanced-stage disorder is reduced or prevented.
  • the symptoms of the skeletal disorder are alleviated.
  • the treatment of the individual results in regression of an advanced-stage skeletal disorder to an early-stage skeletal disorder.
  • the treatment of the individual results in the regression of an advanced-stage skeletal disorder to no skeletal disorder.
  • the method of the invention is intended to encompass all known treatments for skeletal disorders.
  • the skilled person will be familiar with treatments for skeletal disorders.
  • the invention provides a method of determining whether an individual has an early-stage skeletal disorder comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ ⁇ - carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody;
  • MetSO methionine sulfoxide
  • DT dityrosine
  • 3-NT 3-nitrotyrosine
  • test individual has an early-stage skeletal disorder.
  • the method of the invention is for determining whether an individual has an early-stage skeletal disorder. In one embodiment, the method of the invention is for determining whether an individual has an early- stage skeletal disorder selected from: early-stage osteoarthritis, early-stage rheumatoid arthritis and other inflammatory joint disease that may be self-resolving. In one embodiment, the method of the invention is for distinguishing between early-stage skeletal disorders including early-stage osteoarthritis, early-stage rheumatoid arthritis and other inflammatory joint disease that may be self-resolving. In one embodiment, the method of the invention may be for typing an early-stage skeletal disorder in an individual.
  • the typing may involve determining whether the individual has an early-stage skeletal disorder selected from one of: early-stage osteoarthritis, early-stage rheumatoid arthritis and other inflammatory joint disease that may be self-resolving.
  • an early-stage skeletal disorder selected from one of: early-stage osteoarthritis, early-stage rheumatoid arthritis and other inflammatory joint disease that may be self-resolving.
  • the present inventors have demonstrated that by quantifying two specific subsets of oxidised, nitrated and glycated free adducts (or adduct residues), it is possible to determine whether an individual has an early-stage skeletal disorder, and in particular to distinguish between different types of early-stage skeletal disorder.
  • results could be also be improved by including anti-cyclic citrullinated peptide antibody as a marker for early-stage skeletal disorder in the method of the invention.
  • Anti-cyclic citrullinated peptide antibody is a known marker of skeletal health (Raza, K. et al. Predictive value of antibodies to cyclic citrullinated peptide in patients with very early inflammatory arthritis. J.Rheumatol. 32, 231 -238 (2005)). As demonstrated by Example 2 of the application, excellent results are obtained when either of the sub-sets of markers of skeletal health. Good results can, however, still be obtained when using fewer markers.
  • the method for determining whether an individual has an early-stage skeletal disorder comprises quantifying markers of skeletal health that comprise: at least 6 (eg. at least 7, at least 8, at least 9, at least 10, at least 1 1 , or all 12) markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide
  • the markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and anti-cyclic citrullinated peptide antibody.
  • the markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), N £ -carboxymethyl- lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and anti-cyclic citrullinated peptide antibody.
  • MethodSO methionine sulfoxide
  • 3-NT 3-nitrotyrosine
  • FL N e -fructosyl-lysine
  • CML N £ -carboxymethyl- lysine
  • CEL N £ -(1 -carboxyethyl)lysine
  • 3-deoxyglucosone-derived hydroimidazolone 3DG-H
  • the markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody.
  • the combination of markers described herein may additionally comprise the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the method may further comprise an initial step of isolating the oxidised, nitrated, and glycated free adducts from the body fluid sample by ultrafiltration. All embodiments of the ultrafiltration step described above with respect to the method for determining the skeletal health of an individual apply equally to the method for determining whether an individual has an early-stage skeletal disorder.
  • the method may further comprise an initial step of hydrolysing the adduct residues to release amino acids for quantification. All embodiments of the hydrolysis step described above with respect to the method for determining the skeletal health of an individual apply equally to the method for determining whether an individual has an early-stage skeletal disorder.
  • the method of the invention for determining whether an individual has an early-stage skeletal disorder may further comprise the step of quantifying hydroxyproline (Hyp). All embodiments described above with respect to the step of quantifying hydroxyproline (Hyp) in a method of determining the skeletal health of an individual apply equally to the step of quantifying hydroxyproline (Hyp) in a method of determining whether an individual has an early-stage skeletal disorder. By including this additional marker of skeletal health, the overall sensitivity and specificity of the method may be improved. Methods for quantifying hydroxyproline may also involve an initial step of isolating the hydroxyproline by ultrafiltration, as described above.
  • the method of determining whether an individual has an early-stage skeletal disorder further comprises quantifying rheumatoid factor (RF) in a body fluid sample obtained from the test individual.
  • RF rheumatoid factor
  • the method of determining whether an individual has an early-stage skeletal disorder further comprises quantifying citrulline in a body fluid sample obtained from the test individual. Further discussion of suitable methods for detecting citrulline may be found in WO 2014/016584.
  • the method of the invention for determining whether an individual has an early-stage skeletal disorder may further comprise including the age and/or gender of the test individual as further markers. Quantification of gender may for example comprise assigning a value of 1 if the test individual is female, and a value of 0 if the test individual is male.
  • the method of determining whether an individual has an early-stage skeletal disorder comprises the step of "classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm", wherein the diagnostic algorithm is trained on "a population of individuals having known early-stage skeletal disorder”.
  • the "reference value" and/or the “diagnostic algorithm” used in the method of determining whether an individual has an early-stage skeletal disorder is as defined above for the 'method for determining whether an individual has an early-stage skeletal disorder'.
  • the population of individuals used to obtain reference values for the diagnostic algorithm and/or used to train the diagnostic algorithm may comprise at least one individual having early- stage osteoarthritis, at least one individual having early-stage rheumatoid arthritis, and/ or at least one individual having other inflammatory joint disease that may be self-resolving.
  • the population of individuals used to obtain reference values for the diagnostic algorithm may comprise multiple (eg. at least 10) individuals having early-stage osteoarthritis, multiple (eg. at least 10) individuals having early-stage rheumatoid arthritis, and/ or multiple (eg. at least 10) individuals having other inflammatory joint disease that may be self-resolving.
  • the population of individuals comprises at least one individual having early-stage osteoarthritis, at least one individual having early-stage rheumatoid arthritis, and at least one individual having other inflammatory joint disease that may be self-resolving. In one embodiment, the population of individuals comprises multiple (eg. at least 10) individuals having early-stage osteoarthritis, multiple (eg. at least 10) individuals having early-stage rheumatoid arthritis, and multiple (eg. at least 10) individuals having other inflammatory joint disease that may be self-resolving.
  • the population of individuals used as a reference and/or used to train the diagnostic algorithm may additionally comprise at least one individual having no known skeletal disorder. In one embodiment, the population may additionally comprise multiple (eg. at least 10) individuals having no known skeletal disorder. In one embodiment, the population of individuals used as a reference may additionally comprise at least one individual having a known advanced-stage skeletal disorder. In one embodiment, the population may additionally comprise multiple (eg. at least 10) individuals having a known advanced-stage skeletal disorder.
  • the known advanced-stage skeletal disorder may be advanced- stage osteoarthritis. The known advanced-stage skeletal disorder may be advanced-stage rheumatoid arthritis.
  • samples obtained from individuals that have early-stage skeletal disorders have different marker profiles (ie. the abundance of the markers quantified varies between the samples) to those individuals having no skeletal disorder and those individuals having advanced-stage skeletal disorder (including those having advanced-stage OA, and advanced-stage RA).
  • the differences in marker abundance between these individuals provide a way to determine whether an individual has an early- stage skeletal disorder.
  • different marker profiles were also observed for individuals having different early-stage skeletal disorders. The differences in marker abundance between these individuals provide a way to classify individuals as having a particular type of early-stage skeletal disorder, such as early-stage OA, early-stage RA and non-RA.
  • classification of the individual's marker profile as corresponding to the profile derived from a particular reference population is predictive that the patient falls (or does not fall) within the reference population.
  • an individual may be diagnosed as having an early-stage skeletal disorder when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having a known early-stage skeletal disorder. In one embodiment, an individual may be diagnosed as having early-stage OA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having early-stage OA. In one embodiment, an individual may be diagnosed as having early-stage RA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having early- stage RA. In one embodiment, an individual may be diagnosed as having non-RA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having non-RA.
  • the diagnostic algorithm used in the method of the invention is a classification algorithm. All embodiments of the classification algorithm described above with respect to the 'method for determining skeletal health of an individual' apply to the 'method for determining whether an individual has an early- stage skeletal disorder'.
  • the diagnostic algorithm used in the method of the invention for determining whether an individual has an early-stage skeletal disorder is a 3-class algorithm (see Figure 2(b)).
  • the invention further provides the use of at least 6 (eg.
  • markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl- lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), N w -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine and glucosepane; and anti-cyclic citrullinated peptide antibody (anti-CCP antibody) for determining whether a test individual has an early-stage skeletal disorder.
  • MetSO methionine sulfoxide
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), ⁇ ⁇ - carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and anti-cyclic citrullinated peptide antibody (anti-CCP antibody) as markers for determining whether a test individual has an early-stage skeletal disorder.
  • MetalSO methionine sulfoxide
  • DT dityrosine
  • 3-nitrotyrosine 3-NT
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and anti-cyclic citrullinated peptide antibody (anti-CCP antibody) as markers for determining whether a test individual has an early-stage skeletal disorder.
  • MetalSO methionine sulfoxide
  • 3-NT 3-nitrotyrosine
  • FL N £ -fructosyl-lysine
  • CML N £ -carboxymethyl-lysine
  • CEL N £ -(1 -carboxyethyl)lysine
  • the use is of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), ⁇ ⁇ - carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody (anti-CCP antibody) as markers for determining whether a test individual has an early-stage skeletal disorder.
  • MetalSO methionine sulfoxide
  • DT dityrosine
  • the combination of markers described herein may additionally include the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the invention also provides a method of treating an early-stage skeletal disorder in an individual, comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody;
  • the "early-stage skeletal disorder” may be early-stage osteoarthritis (eOA), early- stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving.
  • the method of the invention is intended to encompass all known treatments for early-stage skeletal disorder.
  • the skilled person will be familiar with treatments for early-stage skeletal disorder, such as those for treating early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving.
  • the treatment administered to the individual improves the skeletal health of the individual.
  • the invention also provides a method of determining whether an individual has an early-stage skeletal disorder comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody;
  • the method of determining whether an individual has an early-stage skeletal disorder may comprise:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and hydroxyproline
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and anti-cyclic citrullinated peptide antibody;
  • MetSO methionine sulfoxide
  • 3-NT 3-nitrotyrosine
  • FL N £ -fructosyl-lysine
  • CML ⁇ -carboxymethyl-lysine
  • CEL N £ -(1 -carboxyethyl)lysine
  • the method of determining whether an individual has an early-stage skeletal disorder may comprise:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprise the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and hydroxyproline (Hyp);
  • markers of skeletal health in a body fluid sample obtained from a test individual comprise the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and anti-cyclic citrullinated peptide antibody;
  • MetSO methionine sulfoxide
  • 3-NT 3-nitrotyrosine
  • FL N £ -fructosyl-lysine
  • CML N £ -carboxymethyl-lysine
  • CEL N £ -(1 -carboxyethyl)lysine
  • the method of determining whether an individual has an early-stage skeletal disorder may comprise:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-NT 3-nitroty
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and anti-cyclic citrullinated peptide antibody;
  • the method of determining whether an individual has an early-stage skeletal disorder may comprise:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and hydroxyproline (Hyp);
  • markers of skeletal health in a body fluid sample obtained from a test individual comprise the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), ⁇ ⁇ - carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and anti-cyclic citrullinated peptide antibody;
  • step (ii) classifying the skeletal health based on the amount of each marker quantified in the test sample with a second diagnostic algorithm, wherein the second diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known early-stage skeletal disorder, and thereby determining whether the test individual has an early-stage skeletal disorder.
  • the values obtained for the markers quantified in step (a) part (i) of the method may be used directly in step (c) part (i), thereby avoiding the need to repeat quantification of overlapping markers.
  • step (a) part (i) of the method involves quantification of any one of the oxidised, nitrated and glycated free adducts selected from: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1), pentosidine, and glucosepane, these values may be used directly in step (c) part (i).
  • Methionine sulfoxide MetalSO
  • DT dityrosine
  • 3-nitrotyrosine (3-NT) 3-nitrotyrosine
  • CEL ⁇ ⁇ -(1 - carboxyethyl)lysine
  • CMA ⁇ ⁇ -carboxymethylarginine
  • MG-H1 methylglyox
  • the invention also provides a method of treating an early-stage skeletal disorder in an individual, comprising:
  • markers of skeletal health in a body fluid sample obtained from a test individual comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp), and classifying the skeletal health based on the amount of each marker quantified in
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody, and classifying the skeletal health based on the amount of
  • the "early-stage skeletal disorder” may be early-stage osteoarthritis (eOA), early- stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving.
  • the method of the invention is intended to encompass all known treatments for early-stage skeletal disorder.
  • the skilled person will be familiar with treatments for early-stage skeletal disorder, such as those for treating early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving.
  • eOA early-stage osteoarthritis
  • eRA early-stage rheumatoid arthritis
  • other inflammatory joint disease that may be self-resolving.
  • the treatment administered to the individual improves the skeletal health of the individual.
  • the method for determining the skeletal health of an individual (and for treating a skeletal disorder in an individual), and the method for determining whether an individual has an early-stage skeletal disorder (and for treating an early-stage skeletal disorder in an individual), may further comprise repeating the 'quantification' and 'comparison' steps of the method after a selected time interval and comparing the amount of each marker quantified after said time interval to the amount quantified for each marker at an earlier time point, to determine whether the skeletal health of the test individual has improved, worsened, or remained stable.
  • the method for determining the skeletal health of an individual (and for treating a skeletal disorder in an individual), and the method for determining whether an individual has an early-stage skeletal disorder (and for treating an early-stage skeletal disorder in an individual), may further comprise repeating the 'quantification' and 'classification' steps of the method after a selected time interval and comparing the classification of skeletal health obtained after said time interval to the classification of skeletal health obtained at an earlier time point, to determine whether the skeletal health of the test individual has improved, worsened, or remained stable.
  • the method for determining the skeletal health of an individual (and for treating a skeletal disorder in an individual), and the method for determining whether an individual has an early-stage skeletal disorder (and for treating an early-stage skeletal disorder in an individual), may further comprise repeating the 'quantification' and 'comparison' steps of the method using a body fluid sample obtained from the test individual at one or more later time points and comparing the amount of each marker quantified at the one or more later time points to the amount quantified for each marker at a first (or earlier) time point, to determine whether the skeletal health of the test individual has improved, worsened, or remained stable.
  • the method for determining the skeletal health of an individual (and for treating a skeletal disorder in an individual), and the method for determining whether an individual has an early-stage skeletal disorder (and for treating an early-stage skeletal disorder in an individual), may further comprise repeating the 'quantification' and 'classification' steps of the method using a body fluid sample obtained from the test individual at one or more later time points and comparing the classification of skeletal health determined for the one or more later time points to the classification of skeletal health determined for a first (or earlier) time point, to determine whether the skeletal health of the test individual has improved, worsened, or remained stable.
  • This may be useful for monitoring the skeletal health of a test individual.
  • the method may be useful for monitoring the severity of a skeletal disorder; and/or the effectiveness of a treatment regimen on skeletal health.
  • the disease status of the patient can be re-classified to determine whether there has been a change or no change in the disease status of the patient.
  • the levels of the markers return towards (or becomes increasingly statistically similar to) the levels typically observed for the reference value representative of a healthy individual, and/or increasingly statistically deviates from the level typically observed for the reference value representative of a skeletal disorder (such as a particular type of early stage or advanced stage skeletal disorder), this indicates that there has been an improvement or regression of the skeletal disease in the test individual.
  • the levels of the markers increasingly statistically deviates from the levels typically observed for the reference value representative of a healthy individual, and/or remains statistically similar to (or becomes increasingly statistically similar to) the level typically observed for the reference value representative of a skeletal disorder (such as a particular type of early stage or advanced stage skeletal disorder), this indicates that there has been a worsening or progression of the skeletal disorder in the test individual.
  • Monitoring of the severity of skeletal disorder in a patient may comprise monitoring of the progression, regression, aggravation, alleviation or recurrence of the disorder.
  • Monitoring of the severity of skeletal disorder in a patient may comprise determining whether the skeletal disorder is progressing towards a more advanced form of the disorder, or regressing towards normalcy.
  • Monitoring may also comprise determining whether the skeletal disorder has remained stable.
  • progression refers to an increase or worsening in the symptoms of a disease or disorder
  • regression refers to a decrease or improvement in the symptoms of disease or or disorder
  • Monitoring of the skeletal health in a patient may be used to determine the effectiveness of a treatment regimen on skeletal health.
  • the treatment regimen may include all known treatments (e.g. pharmacological treatments) for early- stage and advantage-stage skeletal disorder.
  • the skilled person will be familiar with treatments for early- stage skeletal disorder, such as those for treating early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or other inflammatory joint disease that may be self-resolving; and for advanced-stage skeletal disorder, such as those for treating advanced-stage osteoarthritis (aOA), advanced -stage rheumatoid arthritis (eaRA).
  • the treatment regimen may include a program of exercise and/or physiotherapy; and/or administration of foods and/or supplements that improve skeletal health.
  • the method for monitoring the skeletal health of a test individual may comprise: (i) quantifying markers of skeletal health in a body fluid sample obtained from a test individual at a first time point, and classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm;
  • step (iii) comparing the classification determined in step (i) to the classification determined in step (ii) to determine whether the skeletal health of the test individual has improved, worsened, or remained stable;
  • the skeletal markers comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3- nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal- derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp), and wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • MetSO methionine sulfoxide
  • NFK N-formy
  • the method for monitoring the skeletal health of a test individual may comprise:
  • step (iii) comparing the classification determined in step (i) to the classification determined in step (ii) to determine whether the skeletal health of the test individual has improved, worsened, or remained stable;
  • the skeletal markers comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone- derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody, and wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known early-stage skeletal disorder. All embodiments described above for the method for determining whether an individual has an
  • the diagnostic algorithm used in the monitoring method may comprise a two-stage classification with a first stage for classification as having or not having a skeletal disorder, and a second stage for classification as having an early-stage skeletal disorder selected from at least one of: early-stage osteoarthritis (eOA), early-stage rheumatoid arthritis (eRA), or self-resolving inflammatory joint disease.
  • the diagnostic algorithm used in the first stage classification may be trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • the diagnostic algorithm used in the second stage classification may be trained on corresponding values for each marker obtained from a population of individuals having known early-stage skeletal disorder.
  • the skeletal markers used in the first stage classification may comprise the markers described above for the method for determining the skeletal health of a test individual.
  • the skeletal markers used in the first stage classification may comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3- nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp).
  • MetSO methionine s
  • the skeletal markers used in the second stage classification may comprise the markers described above for the method for determining whether an individual has an early-stage skeletal disorder.
  • the skeletal markers used in the second stage classification may comprise at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3- nitrotyrosine (3-NT), N e -fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti-cyclic citrullinated peptide antibody.
  • MetSO me
  • the "time interval" of the method may comprise a time period of at least 24 hours (e.g. at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years).
  • at least 24 hours e.g. at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years).
  • the sample obtained from the test individual at the "one or more later time points" may be obtained at least 24 hours (e.g. at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years) after the sample was obtained from the test individual at a first (or earlier) time point.
  • 24 hours e.g. at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years
  • the sample obtained from the test individual at a first (or earlier) time point is obtained from the test individual before or during the course of treatment.
  • the sample may be obtained from the test individual at least 1 hour (e.g. at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 3 weeks, or at least 4 weeks) before treatment.
  • the sample obtained from the test individual at one or more later time points is obtained during or after a course of treatment.
  • the sample may be obtained from the test individual at least 24 hours (e.g. at least 48 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years) after a treatment regimen has begun or has been completed.
  • 24 hours e.g. at least 48 hours, at least 96 hours, at least 120 hours, at least 1 week, at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 12 weeks, at least 16 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 52 weeks, at least 2 years, at least 3 years, or at least 4 years
  • the method comprises quantifying at least one (such as at least 2, 3, 4, 5 or 6) marker selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N- formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), and N £ -fructosyl-lysine (FL).
  • MetSO methionine sulfoxide
  • NFK N- formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine (3-NT)
  • CMA ⁇ ⁇ - carboxymethylarginine
  • FL N £ -fructo
  • the method may comprise classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm, wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • the data presented in Figures 3-8 and 13 demonstrates that markers of skeletal health differ in abundance in advanced-stage and early-stage osteoarthritis.
  • an increase in the abundance of MetSO, NFK, CEL and CMA was observed in advanced-stage osteoarthritis as compared to early-stage osteoarthritis, and a decrease in the abundance of DT and 3-NT was observed in advanced-stage osteoarthritis as compared to early-stage osteoarthritis.
  • the method is performed using blood plasma or blood serum as the body fluid sample, and comprises quantifying at least one (such as at least 2, 3, 4, or 5) marker selected from the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), and ⁇ ⁇ -carboxymethylarginine (CMA).
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine
  • CEL N £ -(1 -carboxyethyl)lysine
  • CMA ⁇ ⁇ -carboxymethylarginine
  • the method is performed using blood plasma or blood serum as the body fluid sample, and comprises quantifying the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3- NT), N £ -(1 -carboxyethyl)lysine (CEL), and ⁇ ⁇ -carboxymethylarginine (CMA).
  • MethodSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine (3- NT)
  • CMA ⁇ ⁇ -carboxymethylarginine
  • the method is performed using synovial fluid as the body fluid sample, and comprises quantifying at least one (such as at least 2) marker selected from the oxidised, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), and N £ -fructosyl-lysine (FL).
  • the method is performed using synovial fluid as the body fluid sample, and comprises quantifying the combination of the oxidised, and glycated free adducts: methionine sulfoxide (MetSO), N- formylkynurenine (NFK), and N £ -fructosyl-lysine (FL).
  • the method involves a step of comparing the amount of the at least one marker quantified in the test sample to a corresponding reference value for the marker obtained from a population of individuals having known skeletal health.
  • the method involves a step of classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm.
  • a diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • an individual may be diagnosed as having early-stage OA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having early-stage OA. In one embodiment, an individual may be diagnosed as having advanced-stage OA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having advanced-stage OA.
  • the population of individuals used to obtain reference values for the diagnostic algorithm and/or used to train the diagnostic algorithm may comprise: at least one healthy individual having no skeletal disorder; at least one individual having early-stage osteoarthritis; and/or at least one individual having advanced-stage osteoarthritis.
  • the population of individuals may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder; multiple (eg. at least 10) individuals having early-stage osteoarthritis; and/or multiple (eg. at least 10) individuals having advanced-stage osteoarthritis.
  • the population of individuals used to obtain reference values for the diagnostic algorithm and/or used to train the diagnostic algorithm may comprise: at least one healthy individual having no skeletal disorder; and at least one individual having advanced-stage osteoarthritis.
  • the population of individuals may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder; and multiple (eg. at least 10) individuals having advanced- stage osteoarthritis.
  • the step of comparing the amount of the at least one marker quantified in the test sample to a corresponding reference value for the marker obtained from a population of individuals having known skeletal health may be performed using a diagnostic algorithm.
  • the step of comparing may be part of a classification algorithm. All embodiments of the diagnostic algorithm described above apply equally to the diagnostic algorithm used in this method.
  • the method of the invention for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis provides a useful way for assessing the effectiveness of treatment for advanced-stage osteoarthritis.
  • the method for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis may be performed to determine whether treatment for advanced-stage osteoarthritis improves the skeletal health of a test individual diagnosed with advanced- stage osteoarthritis.
  • the phrase "determining whether the treatment for advanced-stage osteoarthritis improves the skeletal health of a test individual diagnosed with advanced-stage osteoarthritis" means determining whether the test individual diagnosed with advanced-stage osteoarthritis shows an improvement in their skeletal health as a result of treatment, on the basis of the abundance of markers quantified for the test individual.
  • the body fluid sample may be obtained from a test individual undergoing treatment for advanced-stage osteoarthritis.
  • the body fluid sample may be obtained from the test individual at any stage of the treatment process.
  • the body fluid sample is obtained following at least 1 week (such as at least 2, 4, 8, 12, 20, 30, 40, or 52 weeks, or 2, 3, or 4 years) of treatment for advanced- stage osteoarthritis.
  • the body fluid sample is obtained from the test individual once treatment has been completed.
  • the method may be performed using multiple body fluid samples obtained from the test individual at different time points during the treatment process.
  • the method is performed using a body fluid sample obtained from the test individual prior to commencement of treatment for advanced- stage osteoarthritis, and a body fluid sample obtained from the test individual during treatment (eg. following at least 1 , 2, 4, 8, 12, 20, 40, or 52 weeks, or 2, 3, or 4 years of treatment).
  • the invention further provides the use of the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3- NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), and N £ -fructosyl-lysine (FL) as markers for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis.
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine (3- NT)
  • CMA ⁇ ⁇ -
  • the use is for determining whether treatment for advanced-stage osteoarthritis improves the skeletal health of a test individual diagnosed with advanced-stage osteoarthritis.
  • the invention further provides a method for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis, comprising:
  • the method comprises quantifying at least one (such as at least 2) marker selected from the oxidised, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), and pentosidine. In one embodiment, the method comprises quantifying the combination of the oxidised, and glycated free: methionine sulfoxide (MetSO), dityrosine (DT), and pentosidine.
  • at least one such as at least 2
  • marker selected from the oxidised, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), and pentosidine.
  • the method may comprise classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm, wherein the diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • the method is performed using blood plasma or blood serum as the body fluid sample, and comprises quantifying at least one (such as at least 2) marker selected from the oxidised, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), and pentosidine.
  • the method is performed using blood plasma or blood serum as the body fluid sample, and comprises quantifying the combination of the oxidised, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), and pentosidine.
  • the method is performed using synovial fluid as the body fluid sample, and comprises quantifying at least one marker selected from the oxidised free adduct dityrosine (DT), and the glycated free adduct pentosidine. In one embodiment, the method is performed using synovial fluid as the body fluid sample, and comprises quantifying the combination of the oxidised free adduct dityrosine (DT), and the glycated free adduct pentosidine.
  • DT oxidised free adduct dityrosine
  • DT oxidised free adduct dityrosine
  • the method involves a step of comparing the amount of the at least one marker quantified in the test sample to a corresponding reference value for the marker obtained from a population of individuals having known skeletal health.
  • the method involves a step of classifying the skeletal health based on the amount of each marker quantified in the test sample with a diagnostic algorithm.
  • a diagnostic algorithm is trained on corresponding values for each marker obtained from a population of individuals having known skeletal health.
  • the "reference value” and/or the “diagnostic algorithm” used in the method of determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis is as defined above.
  • an individual may be diagnosed as having early-stage RA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having early-stage RA. In one embodiment, an individual may be diagnosed as having advanced-stage RA when the amount of markers quantified is statistically similar to the amount determined for the corresponding values obtained from a population of individuals having advanced-stage RA.
  • the population of individuals used to obtain reference values for the diagnostic algorithm may comprise: at least one healthy individual having no skeletal disorder; at least one individual having early-stage rheumatoid arthritis; and/or at least one individual having advanced-stage rheumatoid arthritis.
  • the population of individuals may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder; multiple (eg. at least 10) individuals having early-stage rheumatoid arthritis; and/or multiple (eg. at least 10) individuals having advanced-stage rheumatoid arthritis.
  • the population of individuals used to obtain reference values for the diagnostic algorithm and/or used to train the diagnostic algorithm may comprise: at least one individual having early-stage rheumatoid arthritis; and at least one individual having advanced-stage rheumatoid arthritis.
  • the population of individuals may comprise: multiple (eg. at least 10) individuals having early-stage rheumatoid arthritis; and multiple (eg. at least 10) individuals having advanced-stage rheumatoid arthritis.
  • the population of individuals having known skeletal health may comprise: at least one healthy individual having no skeletal disorder; and at least one individual having advanced-stage rheumatoid arthritis. In one embodiment, the population of individuals having known skeletal health may comprise: multiple (eg. at least 10) healthy individuals having no skeletal disorder; and multiple (eg. at least 10) individuals having advanced-stage rheumatoid arthritis.
  • the step of comparing the amount of the at least one marker quantified in the test sample to a corresponding reference value for the marker obtained from a population of individuals having known skeletal health is performed using a diagnostic algorithm.
  • the step of comparing may be part of a classification algorithm. All embodiments of the diagnostic algorithm described above apply equally to the diagnostic algorithm used in this method.
  • the method of the invention for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis provides a useful way for assessing the effectiveness of treatment for advanced-stage rheumatoid arthritis.
  • the method for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis may be performed to determine whether treatment for advanced-stage rheumatoid arthritis improves the skeletal health of a test individual diagnosed with advanced-stage rheumatoid arthritis.
  • the phrase "to determine whether the treatment for advanced-stage rheumatoid arthritis improves the skeletal health of a test individual diagnosed with advanced-stage rheumatoid arthritis” means determining whether the test individual diagnosed with advanced-stage rheumatoid arthritis shows an improvement in their skeletal health as a result of their treatment, on the basis of the of the abundance of markers quantified for the test individual. This means determining whether the abundance of skeletal markers quantified for the test individual is characteristic of an individual having advanced-stage rheumatoid arthritis, or is characteristic of an individual having early-stage rheumatoid arthritis or no skeletal disorder (thereby demonstrating that the test individual has improved skeletal health).
  • the body fluid sample may be obtained from a test individual undergoing treatment for advanced-stage rheumatoid arthritis.
  • the body fluid sample may be obtained from the test individual during any stage of the treatment process.
  • the body fluid sample is obtained following at least 1 week (such as at least 2, 4, 8, 12, 20, 30, 40, or 52 weeks, or 2, 3, or 4 years) of treatment for advanced-stage rheumatoid arthritis.
  • the body fluid sample is obtained from the test individual once treatment has been completed.
  • the method may be performed using multiple body fluid samples obtained from the test individual at different time points during the treatment process.
  • the method is performed using a body fluid sample obtained from the test individual prior to commencement of treatment for advanced- stage rheumatoid arthritis, and a body fluid sample obtained from the test individual during treatment (eg. following at least 1 , 2, 4, 8, 12, 20, 30, 40, or 52 weeks, or 2, 3, or 4 years of treatment).
  • the invention further provides the use of the combination of the oxidised free adducts: methionine sulfoxide (MetSO), and dityrosine (DT), and the glycated free adduct pentosidine as markers for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis.
  • the use is for determining whether treatment for advanced-stage rheumatoid arthritis improves the skeletal health of a test individual diagnosed with advanced-stage rheumatoid arthritis.
  • the invention also provides a kit comprising reagents for quantification of markers of skeletal health, wherein said markers comprise at least 6 (eg.
  • markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal- derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine
  • CEL N £ -
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT); 3-nitrotyrosine (3-NT); ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT); 3-nitrotyrosine (3-NT); ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT); 3-nitrotyrosine (3-NT); ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • NFK N-formylkynurenine
  • DT dityrosine
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine and ⁇ ⁇ -carboxymethylarginine (CMA); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: N-formylkynurenine (NFK), dityrosine (DT), N £ -(1 -carboxyethyl)lysine (CEL), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and
  • markers comprise
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine and N-formylkynurenine (NFK); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: ⁇ ⁇ -carboxymethylarginine (CMA), dityrosine (DT), N £ -(1 -carboxyethyl)lysine (CEL), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL); and
  • markers
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, N w -carboxymethylarginine (CMA) and N-formylkynurenine (NFK); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), ⁇ ⁇ - (l -carboxyethyl)lysine (CEL), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL);
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) and ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), and N e -fructosyl-lysine (FL
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), and methylglyoxal-derived hydroimidazolone (MG-H1); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), and N e -fructosyl-lysine (FL); and
  • markers
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), and dityrosine (DT); and optionally further comprise one or more markers selected from: the glycated free adduct glyoxal-derived hydroimidazolone (G-H1), and N e -fructosyl-lysine (FL); and hydroxyproline (
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), and glyoxal-derived hydroimidazolone (G-H1 ); and optionally further comprise N e -fructosyl-lysine (FL) and/or hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), and N e -fructosyl-lysine (FL); and optionally further comprise glyoxal-derived hydroimidazolone (G-H1) and/or hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), pentosidine, ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK) N £ -(1 -carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), and hydroimidazolone (G-H1); and optionally further comprises the marker hydroxyproline (Hyp).
  • MeSO methionine sulfoxide
  • 3DG-H 3-deoxyglucosone-derived hydroimidazolone
  • 3-nitrotyrosine 3- NT
  • the kit may further comprise reagents for quantification of anti-cyclic citrullinated peptide antibody.
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), and glucosepane (GSP); and may further comprise one or more markers selected from: the oxidised and glycated free adducts: ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal- derived hydroimidazolone (MG-H1), and N £ -fructosyl-lysine (FL); and
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), and ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), and N £ -fructosyl-lysine (FL); and
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), and methylglyoxal-derived hydroimidazolone (MG-H1); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: dityrosine (DT), glyoxal-derived hydroimidazolone (G-H1), and N £ -fructosyl-lysine (FL); and
  • markers comprise
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), and dityrosine (DT); and optionally further comprise one or more markers selected from: the oxidised and glycated free adducts: glyoxal-derived hydroimidazolone (G-H1), and N £ -fructosyl-lysine (FL); and
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), and ⁇ ⁇ - fructosyl-lysine (FL); and optionally further comprise glyoxal-derived hydroimidazolone (G-H1) and/or hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1), dityrosine (DT), and glyoxal-derived hydroimidazolone (G-H1 ); and optionally further comprise N £ -fructosyl-lysine (FL) and/or hydroxyproline (Hyp).
  • MetSO methionine sulfoxide
  • the kit may comprise reagents for quantification of markers of skeletal health, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), 3-nitrotyrosine (3- NT), ⁇ ⁇ -carboxymethylarginine (CMA), N-formylkynurenine (NFK), glucosepane (GSP), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), methylglyoxal-derived hydroimidazolone (MG-H1 ), dityrosine (DT), glyoxal- derived hydroimidazolone (G-H1 ), and N £ -fructosyl-lysine (FL); and optionally further comprises hydroxyproline (Hyp).
  • Methionine sulfoxide MetalSO
  • the kit may additionally comprise reagents for quantification of the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the invention also provides a kit comprising reagents for quantification of markers of early-stage skeletal disorder.
  • the kit may comprise reagents for quantifying the above combinations of markers.
  • the kit may comprise reagents for quantification of markers of early-stage skeletal disorder, wherein said markers comprise at least 6 (eg. at least 7, at least 8, at least 9, at least 10, at least 1 1 , or all 12) markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody.
  • the kit may comprise reagents for quantification of markers of early-stage skeletal disorder, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine; and anti-cyclic citrullinated peptide antibody.
  • the kit may comprise reagents for quantification of markers of early-stage skeletal disorder, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl- lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and glucosepane; and anti-cyclic citrullinated peptide antibody.
  • MetSO methionine sulfoxide
  • 3-NT 3-nitrotyrosine
  • FL N £ -fructosyl-lysine
  • CML N £ -carboxymethyl- lysine
  • CEL N £ -(1 -carboxyethyl)lysine
  • the kit may comprise reagents for quantification of markers of early-stage skeletal disorder, wherein said markers comprise the combination of the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), ⁇ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine and glucosepane; and anti-cyclic citrullinated peptide antibody.
  • the kit may further comprise reagents for quantification of hydroxyproline (hyp).
  • the kit may further comprise reagents for quantification of the oxidised free adducts or adduct residues: AASA and/ or GSA.
  • the invention also provides a kit comprising reagents for quantification of markers for determining whether a test individual has advanced-stage osteoarthritis or early-stage osteoarthritis, wherein said markers comprise: the combination of the oxidised, nitrated and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ -(1 - carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), and N £ -fructosyl-lysine (FL).
  • MeSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine
  • CEL ⁇ ⁇ -(1 - carboxyethyl)lysine
  • the kit comprises reagents for quantification of markers for determining whether treatment for advanced-stage osteoarthritis improves the skeletal health of a test individual diagnosed with advanced-stage osteoarthritis
  • the invention also provides a kit comprising reagents for quantification of markers for determining whether a test individual has advanced-stage rheumatoid arthritis or early-stage rheumatoid arthritis, wherein said markers comprise the combination of the oxidised free adducts: methionine sulfoxide (MetSO), and dityrosine (DT), and the glycated free adduct pentosidine.
  • the kit comprises reagents for quantification of markers for determining whether treatment for advanced-stage rheumatoid arthritis improves the skeletal health of a test individual diagnosed with advanced-stage rheumatoid arthritis.
  • the reference to reagents for quantification of oxidised, nitrated, and glycated free adducts (or adduct residues) and the reference to reagents for quantification of hydroxyproline include a reference to reagents for quantification of their related stable isotype substituted compounds (isotopomers).
  • the reagents for quantification of markers are for quantification of the markers in a body fluid sample obtained from a test individual.
  • the "reagents for quantification of markers” may comprise any reagent that allows the amount of the markers described herein to be determined.
  • the reagents are for quantification of the oxidised, nitrated, and glycated free adducts by isotopic dilution analysis.
  • the reagent for quantifying MetSO is mef ?y/-[ 2 H 3 ]MetSO.
  • the reagent for quantifying Hyp is [ 3 C 2 ]Hyp.
  • the reagent for quantifying NFK is [ 5 N 2 ]NFK.
  • the reagent for quantifying DT is ring-[ 2 H 6 ]Dl .
  • the reagent for quantifying 3-NT is r/ ' r)g-[ 2 H 3 ]3-NT.
  • the reagent for quantifying CEL is /ysy/-[ 3 C 6 ]CEL.
  • the reagent for quantifying CML is /ysy/-[ 3 C 6 ]CML.
  • the reagent for quantifying FL is /ysy/-[ 3 C 6 ]FL.
  • the reagent for quantifying CMA is carboxymethyl-[ C 2 ]CMA.
  • the reagent for quantifying G-H1 is guanidino [ 5 N 2 ]G-H1 .
  • the reagent for quantifying MG-H1 is guan/ ' d/ ' no-[ 5 N 2 ]MG-H1 .
  • the reagent for quantifying 3DG-H is guan/ ' d/ ' no-[ 5 N 2 ]3DG-H.
  • the reagent for quantifying AASA is [ 2 H 3 ]a-Aminoadipic acid.
  • the reagent for quantifying GSA is [ 2 H 3 ]a-Aminoadipic acid.
  • the reagent for quantifying GSP is [ 3 C 6 ]Glucosepane.
  • the reagent for quantifying Pyrraline is [ 3 C 6 , 5 N 2 ]Pyrraline.
  • the reagent for quantifying pentosidine is [ 3 C 6 ]pentosidine.
  • Alternative stable isotopic substitution may be used in these compounds, as may be selected by those skilled in the art of stable isotopic dilution analysis.
  • the reagents for isotopic dilution analysis may comprise at least one (eg. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or all 16) reagents selected from the group consisting of:
  • the reagents may comprise the combination of: [ 3 C 2 ]Hyp, methyl-[ 2 H 3 ] MetSO, [ 5 N 2 ]NFK, ring-[ 2 H 6 ]DT; ring-[ 2 H 3 ]Z-NT; /ysy/-[ 3 C 6 ]CEL, /ysy/-[ 3 C 6 ]CML, /ysy/-[ 3 C 6 ]FL, carboxymethyl- [ 3 C 2 ]CMA, guanidino [ 5 N 2 ]G-H1 , gi/an/d/no-[ 5 N 2 ]MG-H1 , gi/an/d/no-[ 5 N 2 ]3DG-H, [ 2 H 3 ]a-Aminoadipic acid, [ 3 C 6 ]Glucosepane, [ 3 C 6 ]pentosidine and [ 3 C 6 , 5 N 2 ]Pyrraline.
  • kits of the invention may further comprise a known quantity or concentration of the markers described herein (or their related stable isotype substituted compounds (isotopomers)) for use as a standard.
  • kit of the invention may further comprise instructions for carrying out the methods and uses of the invention as described herein.
  • the invention also provides a method for determining the skeletal health of an individual and/or
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1 ), methylglyoxal-derived hydroimidazolone (MG-H1), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine and glucosepane; and hydroxyproline (Hyp);
  • MetSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT dityrosine
  • markers of skeletal health in a body fluid sample obtained from a test individual comprising at least 6 markers selected from: the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), ⁇ ⁇ - fructosyl-lysine (FL), N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1 ), 3- deoxyglucosone-derived hydroimidazolone (3DG-H), pentosidine, and glucosepane; and anti- cyclic citrullinated peptide antibody;
  • the invention also provides a computer program and a computer program product for carrying out any of the methods and uses described herein, and a computer readable medium having stored thereon a program for carrying out any of the methods/ uses described herein.
  • the invention also provides a signal embodying a computer program for carrying out any of the methods and uses described herein, a method of transmitting such a signal, and a computer product having an operating system which supports a computer program for carrying out any of the methods and uses described herein.
  • Figure 1 illustrates compound data bar charts of changes in the levels of oxidised, nitrated, and glycated free adducts and adduct residues of protein in plasma and synovial fluid of patients with early and advanced arthritis.
  • the values used to generate the bar chart are shown in Figures 2-8. Bar chart values are normalised to levels in plasma of healthy control subjects, converted to log 2 scale where zero change is the bar midpoint and horizontal scale range is - 6 - +6.
  • Figure 2 illustrates the training and validation of the diagnostic algorithms for detection of impaired skeletal health and discrimination of early-stage osteoarthritis, rheumatoid arthritis and other inflammatory joint disorders (using the data for "marker group 1 ").
  • ROC curves are given for the training set with AUC and confidence intervals: eOA, 0.98 (0.96 - 1 .00); eRA, 0.91 (0.81 - 1 .00); and non-RA, 0.68 (0.50 - 0.86).
  • a random outcome is 0.33.
  • Figure 3 provides a summary of the data obtained for the oxidation and nitration adduct residues of protein in patient plasma and synovial fluid.
  • Figure 4 provides a summary of the data obtained for the oxidation and nitration free adducts in patient plasma and synovial fluid.
  • Figure 5 provides a summary of the data obtained for lysine-de rived glycation adduct resides in patient plasma and synovial fluid.
  • Figure 6 provides a summary of the data obtained for arginine-derived glycation adduct residues in patient plasma and synovial fluid.
  • Figure 7 provides a summary of the data obtained from lysine-derived glycation free adducts in patient plasma and synovial fluid.
  • Figure 8 provides a summary of the data obtained from arginine derived glycation free adducts in patient plasma and synovial fluid.
  • Figure 9 provides a summary of the homotypic correlation of protein oxidation, nitration and glycation adducts in patient synovial fluid and plasma compartments.
  • Figure 10 provides a summary of the predictive algorithm outcomes for training set cross-validation using Random Forest (based on "marker group 1 ").
  • Figure 1 1 provides a summary of the predictive algorithm outcomes for test set cross-validation using Random Forest (based on "marker group 1 ").
  • Figure 12 provides a summary of the predictive algorithm outcomes for test set validation using Random Forest (based on "marker group 1 ").
  • Figure 13 provides a summary of the data obtained for the glycation free adduct glucosepane and the oxidation free adducts AASA and GSA in patient serum/plasma.
  • Figure 14 provides a summary of the predictive algorithm outcomes for 2-fold cross validation using Support Vector Machines (based on "marker group 2").
  • Figure 15 illustrates (A) MACH-1 mechanical testing system (Biomomentum, Canada); and (B) a view of guinea pig femoral condyle with a position grid superimposed.
  • Figure 16 provides representative pictures of medial compartment of right guinea pig knees of each group over time. Safranin-O/fast green/hematoxylin staining, 4x magnification.
  • Figure 20 illustrates the correlation between the global histological score and OA cartilage thickness or Young modulus.
  • Figure 21 provides a summary of serum concentrations of glycated amino acids in the guinea pig model of osteoarthritis.
  • A FL,
  • B MG-H1 ,
  • C G-H1 ,
  • D CMA,
  • E 3DG-H, and
  • Figure 22 provides a summary of serum concentrations of oxidised and nitrated amino acids in the guinea pig model of osteoarthritis.
  • AASA AASA
  • B GSA
  • C Dityrosine
  • D NFK
  • Figure 24 illustrates the correlation between glycation, oxidation and nitration free adducts and hydroxyproline measured in the guinea pig model of osteoarthritis. Correlations statically significance (P ⁇ 0.05) after a Bonferroni correction of 15 was applied.
  • Figure 25 provides a summary of the levels of plasma glycated, oxidised, nitrated and citrullinated protein quantified in the guinea pig model of osteoarthritis over time.
  • Figure 26 illustrates the correlation between glycated, oxidised, nitrated protein and citrullinated protein in the guinea pig model of osteoarthritis. Correlations statically significance (P ⁇ 0.05) after a Bonferroni correction of 15 was applied.
  • Figure 27 illustrates the correlation of glycation, oxidation and nitration free adducts and citrullinated protein and global histological score in the guinea pig model of osteoarthritis. Correlation coefficients in bold are statistically significant. "Correlation coefficient significant after Bonferroni correction of 16 applied.
  • Figure 28 illustrates the correlation of markers with OA score in the guinea pig model of osteoarthritis.
  • Figure 29 summarises the correlation analysis of glycation, oxidation and nitration free adducts with joint biomechanical properties measured by Mach-1 parameters. "Correlation coefficient significant after Bonferroni correction of 16 applied.
  • EXAMPLE 1 Quantifying levels of oxidised, nitrated and glycated free adducts and adduct residues in samples obtained from healthy individuals and individuals having skeletal disorders
  • aOA advanced OA
  • eOA early-stage OA
  • aRA advanced RA
  • eRA early-stage RA
  • eRA self-resolving inflammatory joint disease
  • eRA early rheumatoid arthritis
  • Criteria for aOA were: longstanding or established severe symptoms of OA (> 2 years duration of disease) with corresponding radiographic changes (Kellgren-Lawrence grade IV changes on plain x-rays) undergoing therapeutic knee aspiration and corticosteroid instillation or total knee replacement.
  • Criteria for aRA were: joint stiffness in the mornings of at least one hour duration; symmetrical swelling in three or more joints; radiographic evidence of bone erosions; rheumatoid nodules with increased serum rheumatoid factor (RF); and symptoms of >2 years duration.
  • Inclusion criteria no history of joint symptoms with no arthritic disorder or other morbidity
  • Exclusion criteria a history of knee injury or knee pain in either knee, taking medication (excepting oral contraceptives and vitamins), and any abnormality at physical examination of the knee.
  • Control subjects and patients with early-stage disease were recruited as two independent cohorts for training set and independent test set for data analysis in machine learning methods.
  • Peripheral venous blood samples were collected with EDTA anti-coagulant from patients pre-operatively and synovial fluid obtained intraoperatively from patients, as appropriate.
  • Peripheral venous blood and synovial fluid were collected and stored and subject characteristics are as previously described (Ahmed, U. et al. Biomarkers of early-stage osteoarthritis, rheumatoid arthritis and musculoskeletal health. Sci. Rep. 5, 9259 (9251 -9257) (2015).
  • Peripheral venous blood samples from healthy subjects and patients with eOA were collected after overnight fasting with EDTA anti-coagulant.
  • Venous blood samples for eRA, non-RA and aOA study groups were collected in the non-fasted state.
  • diurnal variation in serum Hyp from healthy subjects was 20 % and for other amino acids up to 13-25 %, depending on the analyte (Gasser AB, Biological variation in free serum hydroxyproline concentration. Clin Chim Acta. 1980;106(1 ):39-43; Thompson DK, Daily variation of serum acylcarnitines and amino acids. Metabolomics. 2012;8(4):556-65).
  • Plasma samples were centrifuged (2000g, 10 min) and the plasma and synovial fluid supernatant removed and stored at - 80 °C until analysis. Samples were centrifuged within one hour of collection.
  • Ultrafiltrate (50-100 ⁇ ) of plasma/serum or synovial fluid was collected by microspin ultrafiltration (10 kDa cut-off) at 4°C. Retained protein was diluted with water to 500 ⁇ and washed by 4 cycles of concentration to 50 ⁇ and dilution to 500 ⁇ with water over a microspin ultrafilter (10 kDa cut-off) at 4°C. The final washed protein (100 ⁇ ) was delipidated and hydrolysed enzymatically as described, designed and validated to maintain protein oxidation, nitration and glycation adduct content during processing (Rabbani, N., Shaheen, F., Anwar, A., Masania, J. & Thornalley, P.
  • Protein hydrolysate 25 ⁇ , 32 ⁇ 9 equivalent or ultrafiltrate (5 ⁇ ) was spiked with stable isotopic standard analytes, and analysed by LC-MS/MS using an AcquityTM UPLC system with a Quattro Premier tandem mass spectrometer (Waters, Manchester, U.K.)- Samples were maintained at 4°C in the autosampler during batch analysis.
  • the columns were 2.1 x 50 mm and 2.1 mm x 250 mm, 5 ⁇ particle size HypercarbTM (Thermo Scientific), in series with programmed switching, at 30 °C.
  • Chromatographic retention is necessary to resolve oxidised analytes from their amino acid precursors to avoid interference from partial oxidation of the latter in the electrospray ionization source of the mass spectrometric detector.
  • Analytes were detected by electrospray positive ionization, multiple reaction monitoring (MRM) mode where analyte detection response is specific for mass/charge ratio of the analyte molecular ion and major fragment ion generated by collision-induced dissociation in the mass spectrometer collision cell.
  • MRM multiple reaction monitoring
  • the ionization source and desolvation gas temperatures were 120°C and 350°C, respectively.
  • the cone gas and desolvation gas flow rates were 99 and 900 l/h, respectively, and the capillary voltage was 0.60 kV.
  • Argon gas (5.0x10 3 mbar) was in the collision cell.
  • Correction for autohydrolysis of hydrolytic enzymes was made as described (Rabbani, N., Shaheen, F., Anwar, A., Masania, J. & Thornalley, P. J. Assay of methylglyoxal-derived protein and nucleotide AGEs. Biochem.Soc. Trans. 42, 51 1 -517 (2014)).
  • Oxidation adducts MetSO, dityrosine (DT), N-formylkynurenine (NFK), a-aminoadipic semialdehyde (AASA), and glutamic semialdehyde (GSA);
  • Glycation adducts FL, N £ -carboxymethyl-lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ - carboxymethylarginine (CMA), hydroimidazolones derived from glyoxal, methylglyoxal and 3- deoxyglucosone (G-H1 , MG-H1 and 3DG-H), respectively), pentosidine, glucosepane, methylglyoxal-derived lysine dimer (MOLD); and
  • Valine is determined in protein hydrolysates for the protease autohydrolysis correction. Oxidation, nitration and glycation adduct residues are normalised to their amino acid residue precursors and given as mmol/mol amino acid modified; and related free adducts are given in nM. Anti-CCP antibody positivity was assessed by automated enzymatic immunoassay (EliA CCP; Phadia, Uppsala, Sweden).
  • the methods and uses of the invention therefore rely on the patterns of increase and decrease observed in the oxidised, nitrated and glycated free adducts and adduct residues (as discussed in detail below) as indicators of skeletal health in a test individual, so as to determine the skeletal health of the individual, or determine whether the test individual has an early-stage or an advanced-stage skeletal disorder (such as early-stage or advanced-state osteoarthritis or rheumatoid arthritis).
  • an early-stage or an advanced-stage skeletal disorder such as early-stage or advanced-state osteoarthritis or rheumatoid arthritis.
  • the abundance of markers quantified for a test individual is compared to reference values used to train the diagnostic algorithm (such as those obtained for a healthy individual and/ or individuals having known skeletal disorders). Because of the patterns of increase and decrease of markers that are associated with different skeletal conditions (described below), it is possible to determine/classify the skeletal health of the individual.
  • the abundance of markers quantified for the test individual is compared to reference values used to train the diagnostic algorithm (such as those obtained for a healthy individual and/ or individuals having known skeletal disorders). Because of the patterns of increase and decrease observed in markers in early-stage and advanced-stage skeletal disorders (as discussed below), it is possible to determine whether an individual has an early-stage or advanced-stage skeletal disorder.
  • MetSO residue content of plasma protein was increased ca. 2-fold in patients with eOA, eRA, aRA and non-RA and increased ca. 3-fold in patients with aOA, with respect to healthy controls.
  • MetSO residue content of synovial fluid protein was increased 2 to 3 fold in eOA and aOA whereas it was decreased 53% in eRA, with respect to plasma protein content of healthy controls.
  • MetSO residue content of synovial fluid protein of patients with aRA was increased ca. 3-fold with respect to patients with eRA.
  • the increased abundance of MetSO adduct residue observed in the plasma of individuals having early- stage and advanced-stage skeletal disorders as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular in determining whether an individual has a skeletal disorder.
  • the increased abundance of MetSO adduct residue observed in the synovial fluid of individuals having early-stage OA and advanced-stage OA as compared to healthy individuals, and the increased abundance of MetSO observed in the synovial fluid of individuals having advanced-stage RA as compared to individuals having early-stage RA can be used in the methods and uses of the invention as an indicator of skeletal health, e.g. to distinguish between early- stage and advanced-stage RA.
  • NFK residue content of plasma protein was little changed in the study groups except for ca. 3-fold increase in patients with aOA with respect to patient with eOA.
  • the NFK residue content of synovial fluid protein was decreased 85% and 83% in patients with non-RA and eRA, with respect to NFK residue content of plasma protein of healthy controls.
  • the increase observed in the abundance of NFK adduct residue in the plasma of individuals having advanced-stage OA as compared to individuals having early-stage OA can therefore be used in the methods and uses of the invention to distinguish between early-stage and advanced-stage OA.
  • the increase observed in the abundance of NFK adduct residue in the synovial fluid of individuals having advanced-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular as an indicator of advanced-stage OA.
  • DT residue content of plasma protein was increased ca. 55-fold and 56-fold in patients with aRA and aOA whereas it was decreased 68% and 74% in patients with eRA and non-RA, respectively, with respect to healthy controls.
  • DT residue content of plasma protein was markedly increased in advanced versus early- stage disorder.
  • DT residue content of plasma protein was increased ca. 30 fold in aOA with respect to eOA and increased ca.
  • the increased abundance of DT adduct residue in the plasma and synovial fluid of individuals having advanced-stage skeletal disorder as compared to individuals having early-stage skeletal disorder can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. to distinguish between early-stage and advanced-stage skeletal disorders.
  • 3-NT residue content of plasma protein decreased in patients with eOA (-85%), eRA (- 83%) and non-RA (- 80%) but remained unchanged in patients with aOA and aRA, with respect to healthy controls.
  • the 3-NT residue content of protein was decreased 83% in patients with eOA and increased ca. 4-fold in patients with aOA.
  • 3-NT residue content was increased ca. 6-fold in synovial fluid protein, compared to plasma protein, of patients with aOA and eRA - Figure 3.
  • the decrease in the abundance of 3-NT observed in the plasma of individuals having early-stage skeletal disorders as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular as an indicator of early-stage skeletal disorders.
  • the increase in the abundance of 3-NT observed in the synovial fluid of individuals having advanced- stage OA as compared to individuals having early-stage OA can therefore be used in the methods and uses of the invention to distinguish between early-stage and advanced-stage OA.
  • MetSO free adduct concentration of plasma was increased ca. 4-fold in patients with eOA, ca. 6-fold in patients with aOA, ca. 3-fold in patients with eRA, ca. 6-fold in patients with aRA and ca. 4-fold in patients with non-RA, with respect to healthy controls. It was increased ca. 2-fold in advanced disorder, comparing aOA versus eOA and aRA versus eRA.
  • MetSO free adduct concentration of plasma was increased ca. 5-fold in eOA, ca. 15-fold in aOA, ca. 5-fold in eRA, ca. 10-fold in aRA and ca. 4-fold in non-RA, with respect to plasma of healthy controls.
  • the increased abundance of MetSO free adducts observed in plasma and synovial fluid of individuals having early-stage and advanced-stage skeletal disorders as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular in determining whether an individual has a skeletal disorder.
  • the increased abundance of MetSO free adduct observed in the plasma and synovial fluid of individuals having advanced-stage skeletal disorders as compared to individuals having early-stage skeletal disorders can also be used in the methods and uses of the invention to distinguish between early-stage and advanced-stage skeletal disorders.
  • NFK free adduct concentration of plasma was increased ca. 4-fold in patients with aOA, eRA and non- RA, and ca. 5 in patients with aRA, with respect to healthy controls.
  • NFK free adduct concentration of plasma was increased ca. 3-fold in aOA with respect to eOA.
  • concentration of NFK free adduct was increased ca. 2-fold in eOA, ca. 6-fold in aOA, ca. 16-fold in eRA, ca. 5-fold in aRA and ca. 17-fold in non-RA, with respect to plasma of healthy controls.
  • the increased abundance of NFK free adducts observed in plasma and synovial fluid of individuals having early-stage and advanced-stage skeletal disorders as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular in determining whether an individual has a skeletal disorder.
  • the increased abundance of NFK free adduct observed in the plasma and synovial fluid of individuals having advanced-stage OA as compared to individuals having early-stage OA can also be used in the methods and uses of the invention to distinguish between early-stage and advanced-stage OA.
  • DT free adduct concentration of plasma was decreased 60% in patients with aOA, 70% in patients with eRA and 75% in patients with non-RA, with respect to healthy controls. It was decreased 80% in aOA versus eOA whereas it was increased 4-fold in aRA versus eRA.
  • DT free adduct concentration of synovial fluid was increased ca. 2-fold in eOA and aOA and unchanged in other patient study groups, compared to plasma of healthy controls.
  • the decrease in the abundance of DT free adduct in the plasma of individuals having advanced-stage OA as compared to individuals having early-stage OA can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular for distinguishing early-stage and advanced-stage OA.
  • the increase in the abundance of DT free adduct in the plasma of individuals having advanced-stage RA as compared to individuals having early-stage RA can therefore be used in the methods and uses of the invention as an indicator of skeletal health, in particular for distinguishing early-stage and advanced- stage RA.
  • the increase in the abundance of DT free adduct in the synovial fluid of individuals having early-stage and advanced-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health.
  • Plasma 3-NT free adduct concentration was decreased 70% and 64% in plasma and synovial fluid of aOA with respect to healthy controls and unchanged in all other patient groups - Figure 4.
  • the decrease in the abundance of 3-NT free adduct observed in the plasma and synovial fluid of individuals having advanced-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. as an indicator of advanced-stage OA.
  • CML residue content of plasma protein was increased ca. 5-fold in patients with aOA and aRA, with respect to healthy controls. It was increased markedly in advanced disorder: ca. 9-fold increase in aOA versus eOA and aRA versus eRA.
  • a similar effect was found in synovial fluid protein where CML residue content was increased ca. 1 1 -fold in aOA versus eOA and ca. 7-fold in aRA versus eRA.
  • the increase in the abundance of CML adduct residue in the plasma and synovial fluid of individuals having advanced-stage skeletal disorders as compared to individuals having early-stage skeletal disorders can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. for distinguishing early-stage and advanced-stage disorders.
  • CEL residue content of plasma protein was increased ca. 2-fold in patients with eOA and aRA only, with respect to healthy controls.
  • CEL residue content of synovial fluid protein was increased 2-fold with respect to plasma protein in patients with aOA.
  • the increase in the abundance of CEL adduct residue in the plasma of individuals having early-stage OA and advanced-stage RA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health.
  • Pentosidine residue content of plasma protein was increased ca. 23-fold in patients with eOA, ca. 9-fold in patients with aOA and ca. 7-fold in patients with aRA, with respect to healthy controls. Pentosidine residue content of synovial fluid protein was unchanged. In patients with aOA, pentosidine residue content of synovial fluid protein was ca. 7-fold higher than in plasma protein - Figure 5.
  • the increase in the abundance of pentosidine adduct residue observed in the plasma of individuals having early-stage OA, early-stage RA and advanced-stage RA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health.
  • G-H1 residue content of plasma protein was increased ca. 3-fold in patients with aRA, with respect to healthy controls.
  • G-H1 residue content of synovial fluid protein was unchanged, with respect to plasma protein healthy controls.
  • the increase in the abundance of G-H1 adduct residue observed in the plasma of individuals having advanced-stage RA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. as an indicator of advanced-stage RA.
  • MG-H1 residue content of plasma protein was decreased 64% in patients with eRA but increased 2 - 3- fold in patients with aRA; hence it increased ca. 7-fold in aRA versus eRA.
  • MG-H1 residue content of synovial fluid protein was unchanged, with respect to plasma protein healthy controls. It increased ca. 3- fold, however, in aOA versus eOA. There were ca. 4-fold and 2-fold increases in MG-H1 residue content of synovial protein compared to plasma protein in patients with eRA and non-RA, respectively.
  • the increase in the abundance of MG-H1 adduct residue observed in the plasma and synovial fluid of individuals having advanced-stage skeletal disorders as compared to individuals having early-stage skeletal disorders can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. for distinguishing early-stage and advanced-stage disorders.
  • 3DG-H residue content of plasma protein and synovial fluid protein was unchanged, with respect to plasma protein of healthy controls. It was increased, however, ca. 9-fold in plasma protein and ca. 4-fold in synovial protein in aOA with respect to eOA.
  • the increase in the abundance of 3DG-H adduct residue in the plasma and synovial fluid of individuals having advanced-stage OA as compared to individuals having early-stage OA can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. for distinguishing early-stage and advanced-stage OA.
  • CMA residue content of plasma protein was increased ca. 6-fold in aRA, ca. 2-fold in non-RA and was unchanged in synovial fluid protein, with respect to plasma protein of healthy controls. It was increased ca. 4-fold in plasma protein of aOA versus eOA - Figure 6.
  • the increase in the abundance of CMA adduct residue in the plasma of individuals having advanced-stage OA as compared to individuals having early-stage OA can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. for distinguishing early-stage and advanced-stage OA.
  • CEL free adduct concentration in plasma was increased ca. 4-fold in patients with aOA and 2-fold in patients with aRA, with respect to healthy controls.
  • CEL free adduct concentration of synovial fluid of patients with aOA was 61 % lower than in plasma.
  • the increase in the abundance of CEL free adduct observed in the plasma and synovial fluid of individuals having advanced-stage OA as compared to individuals having early-stage OA, and in the plasma and synovial fluid of individuals having advanced- stage RA as compared to individuals having early-stage RA, can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. to distinguish between early-stage and advanced-stage skeletal disorders.
  • MOLD free adduct concentration in plasma was decreased 82% in patients with eOA with respect to healthy controls.
  • MOLD free adduct concentration in synovial fluid was decreased 77% and 68% and in eOA and aOA whereas it was increased ca. 6-fold and 4-fold in eRA and non-RA, with respect to plasma of healthy controls.
  • Pentosidine free adduct concentration in plasma was increased ca. 5-fold in patients with aRA, with respect to healthy controls.
  • synovial fluid it was increased ca. 2-fold in eOA, 3-fold in aOA and 4-fold in aRA, with respect to plasma of healthy controls. It was increased ca. 3-fold in aRA versus eRA in both plasma and synovial fluid - Figure 7.
  • the increase in the abundance of pentosidine free adduct in the plasma and synovial fluid of individuals having advanced-stage OA as compared to individuals having early-stage OA, and in the plasma and synovial fluid of individuals having advanced-stage RA as compared to individuals having early-stage RA can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. for distinguishing early-stage and advanced-stage skeletal disorders.
  • the increase in the abundance of MG-H1 free adduct in the plasma of individuals having advanced-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. as an indicator of advanced-stage OA.
  • the decrease in the abundance of 3DG-H free adduct in the plasma of individuals having early-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. as an indicator of early-stage OA.
  • the increase in the abundance of G-H1 and MG- H1 free adducts in synovial fluid of individuals having early-stage RA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. as an indicator of early-stage RA.
  • the decrease in abundance of 3DG-H free adduct observed in the synovial fluid of individuals having early-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health, e.g. as an indicator of early-stage OA.
  • CMA free adduct concentration in plasma was far more responsive: it increased ca.
  • CMA free adduct concentration increased similarly in synovial fluid - Figure 8.
  • the increase in the abundance of CMA free adduct in the plasma and synovial fluid of individuals having early-stage OA, early-stage RA, non-RA and advanced-stage OA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health.
  • the increase in the abundance of glucosepane in the serum/plasma of indiividuals having all forms of OA and non-RA as compared to healthy individuals can therefore be used in the methods and uses of the invention as an indicator of skeletal health.
  • the increase in the abundance of glucosepane in the serum/plasma of individuals having early-stage RA as compared to individuals having early-stage OA and non-RA can therefore be used in the methods and uses of the invention to distinguish between different types of early-stage skeletal disease.
  • EXAMPLE 2 Generating a predictive diagnostic algorithm for diagnosing early-stage skeletal disorder
  • Two diagnostic algorithms were developed, the first to discriminate between individuals having a skeletal disorder and individuals having no skeletal disorder (e.g. Figure 2(a)), and the second to discriminate between individuals having early-stage OA, early-stage RA and non-RA (e.g. Figure 2(b)).
  • This pair of algorithms could be used separately or in combination to form a two-stage diagnostic testing regime.
  • the data obtained in Example 1 was used to generate diagnostic algorithms for diagnosing skeletal disorder.
  • Various subsets of markers were identified for use in a diagnostic algorithm enabling highly sensitive and specific determination of skeletal health.
  • Example 1 the data obtained in Example 1 for the 12 oxidised, nitrated and glycated free adducts (MetSO, DT, NFK, 3-NT, FL, CML, CEL, C-HI, MG-HI, 3DG-H, CMA and Pentosidine) was used together with data obtained for plasma hydroxyproline, anti-CCP antibodies, and rheumatoid factor (RF).
  • the subject groups used to train the diagnostic algorithms were the healthy control, eOA, eRA and non- RA subject groups. Data were analysed using SPSS, version 22.0, with R version 3.1 .3 used for the diagnostic algorithm analysis.
  • the two predictive algorithms were trained on a training data set, before being used to predict the disorder class for each sample in a test data set.
  • the clinical characteristics of training and test set study groups are as given in Ahmed, U. et al. Biomarkers of early-stage osteoarthritis, rheumatoid arthritis and musculoskeletal health. Sci. Rep. 5, 9259 (9251 -9257) (2015).
  • Machine learning analysis on subject groups with and without early-stage arthritis was performed to assess the predictive power of the measured biomarkers in early-stage skeletal disorder.
  • the training data set used to generate the first diagnostic algorithm for predicting impaired skeletal health was obtained from 52 individuals (including healthy controls, and individuals with impaired skeletal health).
  • the training data set used to generate the second diagnostic algorithm for distinguishing early-stage skeletal disorders (eOA, eRA and non-RA) was obtained from 36 individuals (including individuals with eOA, eRA, and non-RA).
  • Various machine learning algorithms were tested for performance:
  • Random Forests - a nonlinear, tree-based method (Breiman, L. Random Forests. 45, 5-32 (2001)); multi-class logistic regression (GLM));
  • SVM Support Vector Machines
  • ROC curve AUC statistic was used as a measure of performance, with 95% CI determined via bootstrap analysis, using the R package "pROC" (Robin, X. et al. pROC: an open-source package for R and S plus to analyze and compare ROC curves. BMC Bioinformatics 12, 77-85 (201 1)).
  • Example sensitivity/specificity values were produced from the ROC curves via an automated procedure that finds the point on the ROC curve where the sensitivity/specificity values are most similar. The sensitivity and specificity values can however be tuned in other ways.
  • a Random Forest algorithm was trained on the entire training set, before making predictions for the held- out test data set.
  • the predictive algorithm outcomes for training set cross-validation using the Random Forest algorithm are shown in Figure 10.
  • Training set cross-validation was also used to perform a stepwise removal of features that were not improving the mean AUC score. These markers can be omitted without reduction in the training set cross- validation AUC. This reduced feature set was used in the test set validation, but not the training set cross- validation (where it would upwardly bias the result). It was observed that for the disorder-versus-control algorithm, the RF, anti-CCP antibody positivity, CML, and FL markers could be removed without affecting sensitivity and specificity of the algorithm. For the early-disorder-type algorithm, it was observed that the RF, NFK, G-H1 , and Hyp markers could be removed without affecting sensitivity and specificity of the algorithm. The outcome of each analysis was to assign, for each test set sample, a set of probabilities corresponding to each of the disorder/control groups. The predicted group is then the one for which the probability is highest. Testing the algorithm
  • the two predictive algorithms were tested using test data that were held separate from the algorithm training, and no algorithm settings were adjusted after generating the test set results - providing for a rigorous estimate of predictive performance on previously unseen cases.
  • test data set used to test the first diagnostic algorithm for predicting impaired skeletal health was obtained from 129 individuals (including healthy controls, and individuals with impaired skeletal health).
  • the test data set used to test the second diagnostic algorithm for distinguishing early-stage skeletal disorders was obtained from 97 individuals (including individuals with eOA, eRA, and non-RA).
  • the set of markers used in the disorder-versus-control algorithm included a total of 1 1 markers consisting of: hydroxyproline (Hyp), and the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), N-formylkynurenine (NFK), dityrosine (DT); 3-nitrotyrosine (3-NT); N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), glyoxal-derived hydroimidazolone (G-H1), methylglyoxal- derived hydroimidazolone (MG-H1 ), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine.
  • Hyp hydroxyproline
  • MeSO methionine sulfoxide
  • NFK N-formylkynurenine
  • DT
  • the set of markers used in the early-disease-type algorithm included a total of 1 1 markers consisting of: anti-cyclic citrullinated peptide antibody, and the oxidised, nitrated, and glycated free adducts: methionine sulfoxide (MetSO), dityrosine (DT), 3-nitrotyrosine (3-NT), N £ -fructosyl-lysine (FL), N £ -carboxymethyl- lysine (CML), N £ -(1 -carboxyethyl)lysine (CEL), ⁇ ⁇ -carboxymethylarginine (CMA), methylglyoxal-derived hydroimidazolone (MG-H1), 3-deoxyglucosone-derived hydroimidazolone (3DG-H), and pentosidine.
  • MetSO methionine sulfoxide
  • DT dityrosine
  • 3-nitrotyrosine 3-nitrotyrosine (3-
  • the first diagnostic algorithm was able to detect and discriminate early-stage skeletal disorder with test set cross- validation sensitivity/ specificity of 0.89/0.90, and with test set validation sensitivity/ specificity of 0.73/0.73.
  • Area under the curve (AUC) for receiver operating characteristic (ROC) curve was: 0.99; and a random outcome is 0.5.
  • AUC area under the curve
  • ROC receiver operating characteristic
  • the second diagnostic algorithm was able to discriminate between the three early-stage disease types with test set validation sensitivities/specificities of 0.81/0.80 (non-RA), 0.57/0.56 (early RA), 0.83/0.84 (early OA). Mean AUC of ROC curves: 0.86; and a random outcome is 0.33. Testing the algorithm with a reduced set of skeletal markers
  • Algorithms combine those features which improve the diagnostic performance of the test based on increase in area under the curve on the receiver operating characteristic (ROC) curve which relates to the probability of assigning a random sample to the correct clinical group (Hanley JA, McNeil BJ: A method of comparing the areas under receiver operating characteristic curves derived from the same cases. Radiology 1983;148:839-843). It was therefore investigated whether fewer skeletal markers can be used in the predictive algorithm of the invention. By varying the skeletal markers used in the algorithm, it was possible to characterise the loss of diagnostic performance in the predictive algorithm for determining skeletal health.
  • ROC receiver operating characteristic
  • the diagnostic algorithms were trained using a different subset of the markers measured in Example 1 .
  • This subset of markers included: MetSO, DT, NFK, 3-NT, FL, CML, CEL, CMA, G-H1 , MG-H1 , 3DG-H, pentosidine, glucosepane (GSP), glutamic semialdehyde (GSA) and a- aminoadipic semialdehyde (AASA), as well as RF, anti-CCP antibody and Hyp.
  • Support Vector Machines were used to train the algorithm (Sajda P: Machine learning for detection and diagnosis of disease. Annual Review of Biomedical Engineering 2006, 8(1):537-565). For training 50% of the data was used and the remaining 50% data was used as test set for cross validation. The algorithm was validated by 2-fold cross-validation, using 10 randomized repeat trials for improved robustness. A two-stage approach was taken: classification stage (i) to distinguish between disease and healthy control; and classification stage (ii) to distinguish between eOA, eRA and non-RA.
  • the training set cross-validations used a panel of biomarkers consisting of: RF, anti-CCP antibody positivity, Hyp, and free adducts of MetSO, DT, NFK, 3-NT, FL, CML, CEL, CMA, G-H1 , MG-H1 , 3DG-H, pentosidine, glucosepane (GSP), glutamic semialdehyde (GSA) and a-aminoadipic semialdehyde (AASA).
  • GSP glucosepane
  • GSA glutamic semialdehyde
  • AASA a-aminoadipic semialdehyde
  • EXAMPLE 3 Effect of drug therapy on hydroxyproline and protein oxidation, nitration and glycation adducts in plasma and synovial fluid of patients with aRA.
  • Patients receiving anti-TNFa therapy compared to those not receiving anti-TNFa therapy, had lower plasma Hyp (0.96 versus 3.37 ⁇ , P ⁇ 0.01) and MOLD free adduct (43.5 versus 0.5 nM, P ⁇ 0.05) and lower synovial fluid NFK adduct free (43.2 versus 5.6 nM, P ⁇ 0.05), but increased plasma DT free adduct (6.29 versus 4.75 nM, P ⁇ 0.05).
  • Patients receiving treatment with prednisolone compared to those not receiving prednisolone, had: in plasma - higher Hyp (2.87 versus 0.94 ⁇ , P ⁇ 0.05), CML free adduct (230 versus 99 nM, P ⁇ 0.05), 3DG- H, and MetSO protein adduct (0.283 versus 0.016 mmol/mol arg and 47 versus 16 mmol/mol met, respectively, P ⁇ 0.05); and in synovial fluid - lower CEL free adduct (69 versus 453 nM, P ⁇ 0.05), but high 3DG-H free adduct (253 versus 26 nM, P ⁇ 0.05)
  • Treatment with or without methotrexate was associated with increased MG-H1 free adduct in plasma and synovial fluid (plasma: 307 versus 665 nM, P ⁇ 0.05; synovial fluid, 308 versus 829 nM, P ⁇ 0.01) and decreased synovial fluid 3-NT residue content (0.0053 versus 0.0022 mmol/mol tyr, P ⁇ 0.05).
  • Some glycated and oxidised amino acids increased and correlated with the global OARSI histological score, cartilage thickness and Young's modulus.
  • the Dunkin-Hartley guinea pig was used for the experiment. This model is well-characterized for spontaneous knee OA which resembles the development of clinical disease.
  • the attraction of the guinea pig as an OA model system is its histopathological similarity to human disease.
  • This guinea pig OA model is characterized by an early collagen fibril disruption occurring in articular cartilage (2 months). This is followed by the formation of bone cysts (2-3 months), subchondral bone thickening and osteophytes (3-12 months), proteoglycan loss (4-6 months) and fibrillations (8-12 months).
  • the appearance of joint pathology in the guinea pig and human is both age-related and subject to a variety of well-known risk factors, including the weight and mechanical stress. Spontaneous lesions in the knee are balanced and are more pronounced in the medial compartment in the area not covered by the meniscus. This corresponds to the location of lesions in the primary idiopathic OA, in human subjects.
  • Peripheral venous blood samples were collected in the morning at weeks 4, 12, 20 and 28 at the superficial veins of the ears under ketamine (32 mg/kg)/xylazine (3mg/kg) subcutaneous anesthesia. Additional blood samples were collected by intracardiac puncture, under general anesthesia (sodium pentobarbital 200 mg/kg, intraperitoneally) immediately before euthanasia. Blood samples were centrifuged at 2000 g for 5 min, and serum stored at -80°C until analysis.
  • cartilage samples were processed for the histological evaluation.
  • the right knee joint (femoral condyles and tibial plateaus) from each animal was fixed for 24 h in 4 % paraformaldehyde, followed by decalcification in hydrochloric acid (DC2 medium, Labonord, Templemars, France) for 4 h at 4°C before embedding in paraffin.
  • the right kidney and a piece of liver were fixed in 4% paraformaldehyde and included in paraffin.
  • Sections (6 ⁇ ) of the femoral condyles and tibial plateaus were cut with a microtome in the central area not covered by meniscus following the Cushin plane, as recommended by OARSI (Kraus VB, The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the guinea pig. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society. 2010; 18 Suppl 3:S35-52). Three sections at 200 ⁇ of intervals were stained with hematoxylin, fast green and safranin-O, and one supplementary central section was stained with toluidine blue.
  • Each compartment of the section was scored by 2 trained experts blinded from sample identity following OARSI recommendations for the guinea pig model. Briefly, the evaluation considered the cartilage surface integrity (0-8), the proteoglycan content (0-6), the cellularity (0-3), the tidemark integrity (0-1) and the osteophyte (0-3), with a maximum of 21 per compartment. The mean of three sections score were calculated for each knee compartment. To assess the global OA score, scores of each compartment were added, giving a maximal score of 84.
  • the left knee joint (femoral condyles and tibial plateaus) of each animal was used for testing the biomechanical properties of articular cartilage assessed using a Mach-1 ® micromechanical tester (Mach- 1 , Biomomentum Inc., Canada) (Figure 15).
  • a Mach-1 ® micromechanical tester Mach- 1 , Biomomentum Inc., Canada
  • Figure 15 Prior to testing, samples were thawed at room temperature in phosphate buffered saline (PBS) for 30 min to equilibrate before starting the experiment. Subsequently, femoral condyle or tibial plateau was fixed with Loctite ® 4013 glue (Henkel, USA) in a small plastic container. Throughout the testing, each sample was kept moist with PBS.
  • PBS phosphate buffered saline
  • Glycated, oxidised, and nitrated adduct residues and free adducts were measured in the plasma/serum of guinea pigs using stable isotopic dilution analysis LC-MS/MS described above.
  • Plasma Hyp and citrullinated protein in guinea pigs were also measured by stable isotopic dilution analysis LC-MS/MS (Ahmed, U et al.. Biomarkers of early stage osteoarthritis, rheumatoid arthritis and musculoskeletal health. Sci. Rep. 5, 9259 (9251 -9257) (2015)).
  • Results are expressed as mean ⁇ SEM. Following a normality test, a parametric one-way analysis of variance (one-way ANOVA) with Tukey's post-test was performed for histology, MACH-1 and biomarkers (Graphpad Prism 6.0). Statistical significance was represented as p ⁇ 0.05 (*), p ⁇ 0.01 (**), p ⁇ 0.001 (***) or p ⁇ 0.0001 (****). Pearson correlations were performed (GraphPad Prism 6.0) between global OA score, parameters of MACH-1 and biomarkers. For glycated, oxidized and nitrated amino acids, hydroxyproline and serum citrullinated protein analytes analyzed without pre-conceived hypothesis, a Bonferroni correction of 15 is applied.
  • the liver and kidney were examined during euthanasia. No sign of toxicity were observed.
  • the liver and adrenal glands were weighed and no significant differences between the guinea pigs of the same group were observed. Histology
  • MG-H1 , 3DG-H and CML free adducts initially decreases at 12 and 20 weeks, compared to baseline levels, returned to baseline levels at 28 weeks and then increased 2 - 3 fold at 36 weeks.
  • CMA free adduct showed a similar trend except decreasing at 20 and 28 weeks.
  • glucosepane free adduct was unchanged at 12 weeks and then increased progressively at 20, 28 and 36 weeks to 3-fold higher than baseline levels.
  • MetSO, AASA and GSA free adducts were generally increased only at week 36 and by 2 - fold, compared to baseline. Dityrosine and NFK free adducts were increased progressively at weeks 28 and 36 up to 2-fold, compared to baseline.
  • Serum 3-NT concentration was decreased by 29 - 32% at 12 - 36 weeks, compared to baseline.
  • Serum Hyp a bone resorption marker was decreased at weeks 12, 20 and 28, with respect to baseline level (P ⁇ 0.001). See Figures 21 -23 and 25.
  • Homocitrulline levels decreased significantly between week 4 and week 12 (1 .4-fold decrease, p ⁇ 0.05) and increased between week 12 and week 20 (1 .5-fold increase, p ⁇ 0.01).

Abstract

La présente invention concerne un procédé de détermination de la santé squelettique d'un individu comprenant : (a) la quantification de marqueurs de santé squelettique dans un échantillon de fluide corporel obtenu à partir d'un individu d'essai, lesdits marqueurs comprenant au moins 6 marqueurs choisis parmi : les adduits libres oxydés, nitratés et glyqués : sulfoxyde de méthionine (MetSO), N-formylkynurénine (NFK), dityrosine (DT), 3-nitrotyrosine (3-NT), Nε-(1- carboxyéthyl)lysine (CEL), Nω-carboxyméthylarginine (CMA), hydroimidazolone dérivée de glyoxal (G-H1), hydroimidazolone dérivée de methylglyoxal (MG-H1), hydroimidazolone dérivée de 3-désoxyglucosone (3DG-H), pentosidine, et glucosépane ; et hydroxyproline (Hyp); (b) la classification de la santé squelettique sur la base de la quantité de chaque marqueur quantifié dans l'échantillon d'essai avec un algorithme de diagnostic, l'algorithme de diagnostic ayant subi un apprentissage sur des valeurs correspondantes pour chaque marqueur obtenu à partir d'une population d'individus ayant une santé squelettique connue, et ainsi, la détermination de la santé squelettique de l'individu d'essai.
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CN112782292B (zh) * 2020-12-21 2023-04-07 浙江大学医学院附属邵逸夫医院 一种代谢物adma在制备早期骨关节炎诊断试剂盒中的应用
WO2024030649A3 (fr) * 2022-08-05 2024-03-14 Ochalski Pawel G Diagnostics et traitements de syndrome articulaire

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