Chemistry: analytical and immunological testing – Peptide – protein or amino acid – Glycoproteins
Patent
1993-02-11
1994-02-01
Nucker, Christine M.
Chemistry: analytical and immunological testing
Peptide, protein or amino acid
Glycoproteins
530356, 436 86, G01N 33493, C07K 302, C07K 1300
Patent
active
052831973
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a method of monitoring collagen degradation as a diagnostic aid in relation particularly to osteoporosis and rheumatoid arthritis.
Collagen is present in various form in all tissue. It has been shown (Fujimoto et al., (1978) Biochemistry, Biophysics Research Communication vol. 84, 52-57) that collagen has the form of amino acid chains cross-linked by pyridinoline. The pyridinium crosslinks are formed from three hydroxylysine residues, two from the terminal (non-helical) peptides of the collagen molecule that are enzymically converted to aldehydes before reaction and a third hydroxylysine situated in the helical portion of a neighbouring collagen molecule. Robins et al (Annals of the Rheumatic Diseases 1986, 45, 969-973) have described a technique for measurement of pyridinoline in urine by use of an antibody specific to pyridinoline and detected by enzyme-linked immunosorbent assay (ELISA). This technique, however gives a measure only of hydroxylysyl pyridinoline in the sample, and does not recognise lysyl deoxypyridinoline. The former is present in bone and tissue, and the latter in bone only. Thus the technique may indicate the occurrence of collagen degradation, indicating the presence of degenerative disease, but without indicating the type of tissue concerned.
The present invention is based on the recognition that lysyl and hydroxylysyl pyridinoline are present in biological fluid such as urine, attached to fragments of the original amino acid chains, or to sugars. However, there is no certainty as to where the original chains will have broken. In virtually all cases, however, sufficient amino acids will be present to identify the type of tissue from which the particular collagen derived.
Accordingly, the present invention provides a method of monitoring collagen degradation, comprising assaying a biological fluid sample which contains a fragment of collagen including lysyl pyridinoline or hydroxylysyl pyridinoline or a substituted form thereof.
Further according to the present invention there is provided a method of determining the tissue origin of degraded collagen, comprising assaying a biological fluid sample to determine the amount of lysyl pyridinoline or hydroxylysyl pyridinoline containing at least one substituents specific for the origin of the collagen degraded.
From another aspect, the invention resides in an antibody, preferably monoclonal, which is specific to a fragment of collagen which fragment comprises lysyl or hydroxylysyl pyridinoline having at least one amino acid attached thereto.
From another aspect, the invention resides in an antibody, preferably monoclonal, which is specific to a fragment of collagen which fragment comprises hydroxylysyl pyridinoline having a sugar residue attached thereto.
Preferably the pyridinoline is substituted with sections of original amino acid chains.
Preferably, said short sections of amino acid chains each comprise from one to five amino acids.
Preferably the sugar is linked to pyridinoline by glycosylation with galactose or with glucose and galactose.
The collagen may suitably be associated with one or cartilage.
Embodiments of the invention will now be described in further detail by way of example.
Collagen has the general structure: ##STR1## where A's, B's and C's are amino acids and P is lysyl or hydroxylysyl pyridinoline or glycosylated hydroxylysyl pyridinoline. When present in biological fluid, for example, urine, the A, B and C chains are broken and of indeterminate length. The invention is based on the fact that collagen from a specific body tissue will give rise in the biological fluid sample to the presence of ##STR2## where A, B and C are short chains of from one to five amino acids, A, B, and C always being present, and Ax, Ay, Bx, By, Cx, Cy etc. are further parts of the original chains which may or may not be present. Further, A, B and C are specific to a particular tissue of origin. Therefore, this aspect of the invention operates on the unit ##STR3##
In another aspect of the invention X may be a glycosylat
REFERENCES:
patent: 4973666 (1990-11-01), Eyre
Eyre, D., et al., "Identification of Urinary Peptides Derived From Cross-Linking Sites in Bone Collagen in Paget's Disease, " Abstract No. 565 from Jounral of Bone and Mineral Research Jun. 4-7:S210 (1988).
Fujimoto, D., et al., "Analysis of Pyridinoline, a Cross-Linking Compound of Collagen Fibers, in Human Urine," J. Biochem. 94:1133-1136 (1983).
Robins, S. P., Biochem J. 215, 167-173 (1983).
Pinnell, S., Biochim. Biophys. Acta 229, 119-122 (1971).
Dehlinger Peter J.
Nucker Christine M.
Powers Vincent M.
The Rowett Research Institute
Woodward M. P.
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