Method of monitoring collagen degradation

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, a method of determining the tissue origin or degraded collagen thereby.

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 glycosylated hydroxylysyl 
pyridinoline which may or may not have amino acids attached. 
Hydroxylysine residues in the helix are glycosylated by addition of sugar 
groups at their hydroxyl group. Where formation of the pyridinoline (Pyd) 
crosslink involves a glycosylated hydroxylysine, a glycosylated crosslink 
will be produced. The crosslink analogue, lysyl-pyridinoline, formed by 
reaction with a lysine residue in the helix cannot form any glycosylated 
derivatives as it lacks the side-chain hydroxyl group. 
There are two types of glycosylation, either galactose (Gal) alone or the 
disaccharide, glucosyl-galactose (Gal.Glc). Thus, there are two possible 
forms of glycosylated hydroxylysyl pyridinoline as shown below, Pyd-Gal 
and Pyd-Gal.Glc. 
##STR4## 
The relative proportion of mono- to di-saccharide derivatives of 
hydroxylysine varies with different tissues. For the main tissues of 
interest (i.e. those that contain pyridinoline) cartilage contains almost 
entirely Gal.Glc, whereas in bone collagen the monosaccharide 
predominates. 
Both the mono- and di-saccharide derivatives of pyridinoline (structures I 
and II) have been isolated from human urine and identified. These 
components are present normally in urine but have been shown to be present 
in increased amounts in various bone disorders and in arthritic disease. 
The main points of interest in these findings are: 
1. The components are present in urine without hydrolytic treatment and an 
assay procedure would therefore be applicable directly to urine. 
2. Assays of the two components will give some tissue-specific information 
on collagen breakdown. Measurement of Pyd-Gal will provide an index of 
bone collagen resorption: Pyd-Gal.Glc amounts in urine are primarily 
indicative of bone or cartilage degradation, and the relative amounts of 
Pyd-Gal and Pyd-Gal.Glc will provide information on the relative extent of 
bone and cartilage degradation. 
3. The presence of the sugar groups may increase the antigenicity (ease 
with which good antibodies can be raised) of the components. The sugar 
portions will form part of the antibody recognition sites so that 
antibodies specific for each structure can be produced. 
Two types of tissue of particular interest are bone and cartilage. The 
presence of degradation of bone collagen alone is an indicator of 
osteoporosis and other bone disorders, while the presence of degradation 
of both bone collagen and cartilage collagen is an indicator of arthritic 
disorders or diseases. 
In general, the invention can be carried out by following the steps: 
1. Identify a particular X of interest in biological fluid. 
2. Isolate X. 
3. Attach X to a suitable protein. 
4. Inject the product of 3 into a host animal and raise antibody. 
5. Use this antibody in ELISA or other suitable assay technique. 
More specific examples of the invention will now be given. 
Schemes 1 and 2 summarise the strategies for isolating crosslink-containing 
fragments from urine and give details of the components being isolated. 
Variations on these Schemes are detailed below.

EXAMPLE 1 
Method 
a. Urine samples containing high concentrations of total pyridinium 
crosslinks were collected from patients with disorders involving either 
increased bone turnover (hyperparathyroidism) or increased cartilage and 
bone degradation (rheumatoid arthritis). 
b. A total of 1.0-1.51 of urine was freeze-dried, redissolved in 0.2 M 
acetic acid and was subjected to gel filtration chromatography in batches 
using a 2.6.times.140cm column of Biogel P2. 
c. Selected fractions containing pyridinium crosslink derivatives were 
re-chromatographed by reversed-phase HPLC using a C.sub.18 support and 
elution with a mobile phase containing acetonitrile. The peptides were 
further purified by ion-exchange HPLC using DEAE- and SP-5PW columns. 
d. The isolated components were characterized by fast atom bombardment mass 
spectrometry and amino acid sequence analysis; glycosylation sites were 
identified by gas-liquid chromatography of alkali hydrolysates of the 
peptides. 
e. With a knowledge of the amino acid sequences around the crosslink sites 
in bone and cartilage, the tissues of origin of the peptides isolated from 
urine were established. A number of three peptides containing the core 
sequence representative of different tissues was chosen for raising 
antibodies. 
f. Each of the purified peptides (1 .mu.mol) were covalently attached to 
ovalbumin (0.25 .mu.mol) using N-ethyl-N'-(3-dimethylaminopropyl) 
carbodiimide hydrochloride. 
g. Balb/c mice were immunized with the ovalbumin conjugates and monoclonal 
antibodies were produced after fusion with Ag8.653 myeloma cells. Positive 
clones were detected by ELISA using microtube plates coated with the 
peptides attached to gelatin by reaction with a different carbodiimide 
reagent N-cyclohexyl-N'-2-(4'-methylmorpholinium) ethyl 
carbodiimide-p-toluene sulphonate. 
The monoclonal antibodies so produced may be used in immunoassay tests for 
the diagnosis and monitoring of various types of bone disorders and 
degenerative joint diseases. 
Example 2 
Method 
Step 1. Urine (300ml), concentrated to one tenth its original volume, was 
chromatographed on a column (3.0.times.100cm ) of sephadex G25M with 
0.2M-acetic acid as eluant. The fractions between 540 and 630ml (V.sub.e 
/V.sub.o =1.6-1.9) that contained approx. 60% of the characteristic 
pyridinium crosslink fluorescence were pooled and freeze-dried. 
Step 2. The fraction from Step 1 (280mg) was re-chromatographed on a column 
(1.7.times.140cm) of Sephadex G10 eluted with 0.2M-acetic acid to separate 
the crosslinking components from amino acids and other small-M material. 
The fractions were monitored by fluorescence (Ex 295nm; Emm 400nm) and 
material that eluted between 105 and 121ml (V.sub.e /V.sub.o =1.1-1.3) was 
pooled and evaporated to dryness. 
Step 3. The fraction from Step 2 was subject to cation-exchange 
chromatography on a column (0.9.times.15cm) of sulphonated polystyrene 
resin beads (5.mu.) crosslinked (8%) with divinyl benzene (Locarte Co Ltd, 
London; Cat No LA 48/08) run at 56 C and eluted with 67mM sodium citrate 
buffer, pH 3.84. Fractions of 5ml were collected and appropriate fractions 
were desalted on a column (1.7.times.50cm) of Sephadex GIO and 
freeze-dried. 
In this system, Pyd-Gal (I) eluted between 91 and 105min and pyd-Gal.Glc 
(II) eluted between 41 and 50min. 
Final purification of I and II was reversed-phase HPLC on a column 
(0.9.times.25cm) of Rosil C.sub.18 packing (3.mu.), eluted with 0.1% 
heptafluorobutyric acid and a linear gradient from 15 to 30% acetonitrile 
over 30 min. (I) and (II) eluted at 17.3 and 16.9min respectively. 
CHARACTERISATION 
1. Hydrolysis characteristics 
Mild acid hydrolysis (0.1 M HCI at 108 C for 12h) converted Pyd-Gal.Glc 
(II) completely into a mixture of Pyd-Gal (I) and pyridinoline (Pyd). 
Hydrolysis of Pyd-Gal (I) in 2M HCI at 108 C for 8h effected complete 
conversion of this component to Pyd. This behaviour is characteristic for 
these types of compound and has been noted previously for the 
interconversion of hydroxylysine glycosides to hydroxylysine. 
2. Composition 
On hydrolysis with strong mineral acid, both compounds (I) and (II) 
produced Pyd with no other amino acids detected in the hydrolysates. The 
carbohydrate compositions were determined by gas-liquid chromatography of 
methylated derivatives, after hydrolysis in 2M H.sub.2 SO.sub.4 and 
reduction with sodium borohydride Based on these results and determination 
of the amounts of crosslink by HPLC, the molar ratios were: 
Component I Pyridinoline (1.0); Galactose (0.9) 
Component II Pyridinoline (1.0): Galactose (0.8); Glucose (0.9) 
The results above therefore confirm that the structures of compounds I and 
II are as shown earlier. 
In addition to the above, an associated derivative, termed `X`, that eluted 
between 184 and 195min at Step 3, has been isolated. This component 
contains Pyd and is increased in amount in patients with bone disorders. 
Immunoassays for these components will be developed precisely as described 
for the peptides by conjugation to ovalbumin through their amino or 
carboxyl groups. 
Modification and improvements may be incorporated without departing from 
the scope of the invention. 
##STR5##