Note: Descriptions are shown in the official language in which they were submitted.
13479
-1-
ASSAY FOR IN VITRO QUANTITATIVE MEASURMENT
OF CARBOXY-TELEOPETIDES OF TYPE I COLLAGEN
This invention relates to a methodfor assaying bone resorption rates. More
specifically, it relates
to a method for quantitating specific urinary cross-linking amino acids, and
peptide fragments that contain
those amino acids derived from degraded bone collagen.
Bacl~round of the Invention
Osteoporosis isthemostcommonbonediseaseinman. Primary osteoporosis, with
increased
susceptibility to fractures, results from aprogressive net loss of skeletal
bone mass. It is estimated to affect
15-20 million individuals in the United States. Its basis is an age-dependent
imbalance in bone
remodelling, i.e., inthe rates of synthesis and degradation of bone tissue.
About 1.2 million osteoporosis-
related fractures occur in the elderly each year, including about 538,000
compression fractures of the
spine, about 227,000 hip fractures, and a substantial number of early
fracturedperipheral bones. Twelve
to 20% of the hip fractures are fatal because they cause severe trauma and
bleeding, and half of the
surviving patients requirenursing homecare. Total costs fromosteoporosis-
related injuries now amount
to atleast$7billionannually (Barnes, O.M., Science, 236,914 (1987)).
Osteoporosis is most common
in postmenopausal women who, on average, lose 15% of their bone mass in the 10
years after menopause.
This desease also occurs in men as they get older and in young amenon heic
women athletes. Despite the
major, and growing, social and economic consequences of osteoporosis, no
method is available for
measuring bone resorption rates in patients or normal subjects. A major
difficulty in monitoring the disease
is the lack of a specific assay for measuring bone resorption rates.
Methods for assessing bone mass often rely on measuring whole-body calcium by
neutron
activation analysis or mineral mass in a given bone by photon absorption
techniques. These measurements
can give only long-term impressions of whether bone mass is decreasing.
Measuring calcium balances by
comparing
X
13~~~1~
-2-
intake with output is tedious, unreliable, and can only indirectly appraise
whether
bone mineral is being lost over the long term. Other methods currently
available
for assessing decreased bone mass and altered bone metabolism include quantita-
tive scanning radiometry at selected bone locations (wrist, calcaneus, etc.)
and
histomorphometry of iliac crest biopsies. The former provides a crude measure
of
the bone mineral content at a specific site in a single bone. Histomorphometry
gives a semi-quantitative assessment of the balance between newly deposited
bone
seams and resorbing surfaces.
A urinary assay for the whole-body output of degraded bone in 24 hours
would be much more useful. Mineral studies (e.g., calcium balance) cannot do
this
reliably or easily. Since bone resorption involves degradation of the mineral
and
the organic matrix, a specific biochemical marker for newly degraded bone
products in body fluids would be the ideal index. Several potential organic
indices
have been tested. For example, hydroxyproline, an amino acid largely
restricted
to collagen, and the principal structural protein in bone and all other
connective
tissues, is excreted in urine. Its excretion rate is known to be increased in
certain
conditions, notably Paget's disease, a metabolic bone disorder in which bone
turnover is greatly increased. For 'this reason, urinary hydroxyproline has
been
used extensively as an amino acid marker for collagen degradation. Singer,
F.R.,
et al. (1978) In: Metabolic Bone Disease, Vol. II (eds. Avioli, L.V. and
Krane, S.M.)
pp. 489-575, Academic Press, New York.
Goverde (U.S. Patent No. 3,600,132) discloses a process for determination of
hydroxyproline in body fluids such as serum, urine, lumbar fluid, and other
intercellular fluids in order to monitor deviations in collagen metabolism. In
particular, this inventor notes that in pathologic conditions such as Paget's
disease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in
collagen tissues, and in various forms of dwarfism, increased collagen
anabolism
or catabolism as measured by hydroxyproline content in biological fluids can
be
determined. This inventor measures hydroxyproline by oxidizing it to a pyrrole
compound with hydrogen peroxide and N-chloro-p-toluenesulphonamide followed
by colorimetric determination in p-dimethyl-amino-benzaldehyde.
In the case of Paget's disease, the increased urinary hydroxyproline probably
comes largely from bone degradation, hydroxyproline, however, generally cannot
be used as a specific index. Much of the hydroxyproline in urine may come from
new collagen synthesis (considerable amounts of the newly made protein are
degraded and excreted without ever becoming incorporated into tissue fabric),
and
from turnover of certain blood proteins as well as other proteins that contain
-3- 130?~~
hydroxyproline. Furthermore, about 8096 of the free hydroxyproline derived
from
protein degradation is metabolized in the liver and never appears in the
urine.
Kiviriko, K.I. (1970) Int. Rev. Connect. Tissue Res. 5, 93, and Weiss, P.H.
and
Klein, L. (1969) J. Clin. Invest. 48, 1.
Hydroxylysine and its glycoside derivatives, both peculiar to collagenous
proteins, have been considered to be more accurate than hydroxyproline as
markers of collagen degradation. However, for the same reasons described above
for hydroxyproline, hydroxylysine . and its glycosides are probably equally
non
specific markers of bone resorption. Krane, S.M. and Simon, L.S. (1981)
Develop.
Biochem. 22, 185.
In addition to amino acids unique to collagen, various non-collagenous
proteins of bone matrix such as osteocalcin, or their breakdown products, have
formed the basis of immunoassays aimed at measuring bone metabolism. Price,
P.A. et al. (1980) J. Clin. Invest. 66, 878, and Gundberg, C.M. et al. (1984)
Meth.
Enzymol. 107, 516. However, it is now clear that bone-derived non-collagenous
proteins, though potentially a useful index of bone metabolic activity, are
unlikely
on their own to provide quantitative measures of bone resorption. The
concentration in serum of osteocalciii, for example, fluctuates quite widely
both
normally and in metabolic bone disease. Its concentration is elevated in
states of
high skeletal turnover, but it is unclear whether this results from increased
synthesis or degradation of bone. Krane, S.M., et al. (1981) Develop. Biochem.
22,
185, Price, P.A. et al. (1980) J. Clin. Invest. 66, 878, and Gundberg, C.M. et
al.
(1984) Meth. Enzymol. 107, 516.
Collagen Cross-Linking
The polymers of most genetic types of vertebrate collagen require the
formation of aldehyde-mediated cross-links for normal function. Collagen alde-
hydes are derived from a few specific lysine or hydroxylysine side-chains by
the
action of lysyl oxidase. Various di-, tri-, and tetrafunetional cross-linking
amino
acids are formed by the spontaneous intra- and intermolecular reactions of
these
aldehydes within the newly formed collagen polymers; the type of cross-linking
residue varies specifically with tissue type (see Eyre, D.R. et al. (1984)
Ann. Rev.
Biochem. 53: 717-748). Two basic pathways of cross-linking can be
differentiated
for the banded (67nm repeat) fibrillar collagens, one based on lysine
aldehydes, the
other on hydroxylysine aldehydes. The lysine aldehyde pathway dominates in
adult
skin, cornea, sclera, and rat tail tendon and also frequently occurs in other
soft
connective tissues. The hydroxylysine aldehyde pathway dominates in bone,
cartilage, ligament, most tendons, and most internal connective tissues of the
-4-
body, Eyre, D.R. et al. (1974) vida supra. The operating pathway is governed
by
whether lysine residues are hydroxylated in the telopeptide sites where
aldehyde
residues will later be formed by lysyl oxidase (Barnes, M.J. et al. (1974)
Biochem.
_J. 139, 461). The chemical structures) of the mature cross-linking amino
acids on
the lysine aldehyde pathway are unknown, but hydroxypyridinium residues have
been identified as mature products on the hydroxylysine aldehyde route. On
both
pathways and in most tissues, the intermediate, borohydride-reducible cross-
linking residues disappear as the newly formed collagen matures, suggesting
that
they are relatively short-lived intermediates (Bailey, A.J. et al. (1971) FEBS
Lett.
16, 86). Exceptions are bone and dentin, where the reducible residues persist
in
appreciable concentration throughout life, in part apparently because the
rapid
mineralization of the newly made collagen fibrils inhibits further spontaneous
cross-linking interactions (Eyre, D.R. (1981) In: The Chemistry and Biology of
Mineralized Connective Tissues (Veis, A. ed.) pp. 51-55, Elsevier, New York,
and
Walters, C. et al. (1983) Calc. Tiss. Intl. 35: 401-405).
Two chemical forms of 3-hydroxypyridinium cross-link have been identified
(Formula I and II). Both compounds are naturally fluorescent, with the same
characteristic excitation and emissioA spectra (Fujimoto, D. et al. (1977)
Biochem.
Biophys. Res. Commun. 76, 1124, and Eyre, D.R. (1981) Develop. Biochem. 22,
50). These amino acids can be resolved and assayed directly in tissue
hydrolysates
with good sensitivity using reverse phase HPLC and fluorescence detection.
Eyre,
D.R. et al. (1984) Analyt. Biochem. 137: 380-388.
T~IIRMTTT.e r FORMtII,A II
/ NH
iC\NH2 iC~ 2
H CH2 COOH H N CH2 COOH
2~CH-CHZ CH2 ~ ~ "'OH 2 ~H--CH2 CH2 ~ \ OH
HOOCH N J HOOC +/~
N
IH2 H2
H-OH ~H
12
H2 C(H2
~H2 ~H2
C~ ~H
H2N~ COOH H2N ~COOH
hydroxylysyl pyridinoline (HP) lysyl pyridinoline (LP)
1~~~~1~9
-5-
In growing animals it has been reported that these mature cross-links may be
concentrated more
in
anunmineralizedfractionofbonecollagenthaninthemineralizedcollagen(Banes,A.J.,et
al. (1983)
Biochem. Biophys. Res. Commun.113,1975). However, other studies on young
bovine or adult human
bone do not support this concept, Eyre, D.R. (1985) In' The Chemistry and
Biology of Mineralized
Tissues (Butler, W.T. ed.) p 105, Ebsco Media Inc., Birmingham, Alabama.
The presence of collagen hydroxypyridinium cross-links in human urine was
first reported by
Gunja-Smith andBoucek (Gunja-Smith, Z. and Boucek, R.J. (1981) BiochemJ.197:
759-762) using
lengthy isolationprocedures forpeptides and conventional amino acid analysis.
Atthat time, they were
awareonly of the HP formof the cross-link. Robins (Robins, S.P. (1982)
BiochemJ. 207: 617-620) has
reported anenzyme-linked immunoassay to measure HP in urine, having
raisedpolyclonal antibodies to
the free amino acid conjugated to bovine serum albumin. This assay is intended
to provide an index for
monitoring increased joint destruction that occurs with arthritic diseases and
is based, according to Robins,
on the finding that pyridinoline is much more prevalent in cartilage than in
bone collagen. In more recent
work involving enzyme-linked immunoassay, Robins reports that lysyl
pyridinoline is unreactive toward
antiserum to pyridinoline covalently linked to bovine serum albumin (Robins et
al. (1986) Ann. Rheum.
Diseases 45, 969-973). Robins' urinary index for cartilage destruction is
based on the discovery that
hydroxylysyl pyridinoline, derivedprimarily from cartilage, is found in urine
at concentrations proportional
to the rate of joint cartilage resorption. In principal, this index could be
used to measure whole body
cartilage loss; however, no information on bone resorption would be available.
Indeed, it seems more
likely that Robin's assays were largely measuring the increased bone
remodelling that occurs in rheumatoid
arthritis rater than cartilage destruction.
A need therefore exists for a method that allows the measurment of whole-body
bone resorption
rates in humans. The most useful such method would be one that could be
applied to body fluids,
especially urine. The method should be sensitive, i.e., quantifiable down to 1
picomole, and rapidly
measure 24-hour bone resorption rates so that the progress of various
therapies (e. g., estrogen) can be
assessed.
X
-Sa-
SUMMARY OF THE INVENTION
It is an obj ect of the present invention to provided an ass ay for in-vitro
quantitative measurement
of carboxy-telopeptides of type I collagen. In accordance with an aspect of
the present invention there
is provided a method of analyzing a body fluid sample for the presence of an
analyte indicative of type I
collagen degradation in vivo, comprising the steps of contacting the body
fluid sample with an
immunological binding partner which is capable of binding to the analyte,
detecting any binding of the
immunological binding partner in the body fluid sample, and correlating the
detected binding to type I
collagen degradation in vivo, wherein the immunological binding partner is
capable of binding to a peptide
containing a 3-hydroxypyridinium cross-link derived from the carboxy-terminal
telopeptide domain of type
I collagen, the peptide comprising two amino acid sequences derived from the
carboxy-terminal
telopeptide domain of the al (I) chain of type I collagen.
In accordance with another aspect of the present invention there is provided a
test kit for analyzing
a body fluid sample for the presence of an analyte indicative of type I
collagen degradation in vivo,
comprising an immunological binding partner which binds to a peptide derived
from type I collagen
resorption and having a 3-hydroxypyridinium cross-link, the peptide fragment
having two amino acid
sequences derived from the carboxy-terminal telopeptide domain of the al (I)
chain of type I collagen.
In accordance with another aspect of the present invention there is provided
an enzyme linked
immunosorbent assay (ELISA) kit for the in vitro quantitativemeasurementof
carboxy-telopeptides of
type I collagen in urine as an indicatorof human bone resorption, comprising a
polyclonal antibody specific
for carboxy-telopeptide degradation products of type I collagen, wherein the
polyclonal antibody is
characterized by binding to a peptide derived from the carboxy-terminal
telopeptide domain of type I
collagen and having 3-hydroxypyridinium cross-
._ 1~~~~~9~
-5b-
link, and wherein the peptide is isolatable from a urine sample of apatient
with active Paget's disease by
a process which comprises the following steps:
a) dialyzing the urine sample in reduced porosity dialysis tubing (>3,500),
and freeze-drying
the non-diffusate;
b) chromatographing the dried material on a Bio-Gel P2 column in 10% acetic
acid at room
temperature, identifying a region of effluent that contains 3-
hydroxypyridinium cross-linked
peptides by measuring the flourescence of collected fractions at 297 nm
excitation/395
nm emission, and freeze-drying the pooled flourescent fractions;
c) chromatographing the dried material on a Bio-Gel P-4 column eluted at 10%
acetic acid,
identifying two overlapping fraction pools by flourescence of the eluant as
measured
above, and freeze-drying the earlier fraction pool;
d) chromatographing the dried material by ion-exchange HPLC on a TSK DEAE-5-PW
column eluted with a gradient of NaCI (0-0.2M) in 0.02M Tris-HCI, pH 7.5
containing
10% (v/v) acetonitrile, identifying carboxy-terminal type I collagen
telopeptide peaks that
elute between 0.08M and 0.15M NaCI by fluorescence as measured above, and
freeze-
drying each peak;
e) chromatographing each driedpeak on a C-18 reverse phase HPLC column eluted
with
agradient (0-10%) of acetonitrile:n-propanol (3:1 v/v)
in0.OlMtriflouroaceticacidto
isolate the individual peptides;
f) selecting isolatedpeptides that contain a 3-hydroxypyridinium cross-link
derivedform the
carboxy-terminal telopeptide domain of type I collagen; and
."'.:
~~4~,~~~
-5c-
g) confirming that the polycyclonal antibody binds to at least one of the
isolated peptides.
In accordance with another aspect of the present invention there is provided
an enzyme linked
immunosorbent assay (ELISA) kit for the quantification of degradation products
of carboxy-terminal
telopeptides of type I collagen in human serum, comprising an enzyme conj
ugated monoclonal antibody
specific for degradation products of carboxy-terminal telopeptides of type I
collagen, wherein the
monoclonal antibody is characterized by binding to a peptide derived from type
I collagen resorption and
having a 3-hydroxpyridinium cross-link, the peptide having two amino acid
sequences derived from the
carboxy-terminal telopeptide domain of the al (I) chain of type I collagen.
In accordance with another aspect of the present invention there is provided a
radioimmunoassay
(RIA) kit for the quantitative determination of type I collagen specific
sequence in human urine for
monitoring bone resorption, comprising a radionuclide conjugated monoclonal
antibody specific for
degradation products of carboxy-terminal telopeptides of type I collagen,
wherein the monoclonal antibody
is characterized by binding to a peptide derived from type I collagen
resorption and having a 3-
hydroxypyridinium cross-link, the peptide having two amino acid sequences
derived from the caroxy-
terminal telopeptide domain of the al (I) chain of type I collagen.
A method for determining the absolute rate of bone resorption comprising
quantitating the
concentrationof peptide fragments having 3-hydroxypyridinium cross-links
derived from bone collagen
resorption in a body fluid is provided. The
x
1~~.~~~9
-s-
quantitating steps consists of contacting the body fluid with an immunological
binding partner specific to a peptide fragment having 3-hydroxypyridinium
cross-
links derived from bone collagen resorption. In one embodiment of the
invention,
the body fluid is optionally purified prior to the contacting step. This
purification
step is selected from a number of standard procedures, including cartridge
adsorp-
tion and elution, molecular sieve chromatography, dialysis, ion exchange,
alumina
chromatography, hydroxyapatite chromatography, and combinations thereof.
The invention also encompasses other methods for quantitating the concen
tration of peptide fragments having 3-hydroxypyridinium cross-links in a body
fluid. These methods include electrochemical titration, natural fluorescence
spectroscopy, and ultraviolet absorbance. Electrochemical titration may be
conducted directly upon a body fluid without further purification. However,
when
this is not possible due to excessive quantities of contaminating substances,
the
body fluid is first purified prior to the electrochemical titration step.
Suitable
t5 methods for purification prior to electrochemical detection include
dialysis, ion
exchange chromatography, alumina chromatography, molecular sieve
chromatography, hydroxyapatite chromatography, and ion exchange absorption and
elution.
Fluorometric measurement of a body fluid containing 3-hydroxypyridinium
cross-links is an alternative way of quantitating bone resorption. The
fluorometric assay can be conducted directly on a body fluid without further
purification. However, for certain body fluids, particularly urine, it is
preferred
that purification of the body fluid be conducted prior to fluorometric assay.
This
purification step consists of dialyzing an aliquot of urine against an aqueous
solution, thereby producing partially purified peptide fragments retained
within
the nondiffusate. The nondiffusate is then lyophylized, dissolved in an ion
pairing
solution and absorbed onto an affinity chromatography column. The
chromatography column is washed with a volume of ion pairing solution, and,
thereafter, the peptide fragments are eluted from the column with an eluting
solution. These purified peptide fragments are then hydrolyzed and the
hydrolyzate is resolved chromatographically. Chromatographic resolution is
conducted by either high-performance liquid chromatography or microbore high
performance liquid chromatography.
The invention includes a peptide fragment derived from bone collagen
substantially free from other human peptides obtained from a body fluid. The
peptide fragment may contain 3-hydroxypyridinium cross-links; in particular,
lysyl
pyridinoline cross-links and hydroxylysyl pyridinoline cross-links.
-?- 1~~~~199
A specific peptide fragment having a 3-hydroxpyridinium cross-link derived
from the aminoterminal telopeptide domain of bone type I collagen has the
following amino acid sequence.
Asp-Glu-K-Ser-Thr-Gly-Gly.
m
Gln-Tyr-Asp-Gly-K-Gly-Val-Gly
K
where K
t
K
i
K
is hydroxylysyl pyridinoline or lysyl pyridinoline and,
Gln is glutamine or wholly cyclized pyrrolidone carboxylic acid.
The invention also encompasses a peptide fragment containing
3-hydroxypyridinium cross-links derived from the carboxyterminal telopeptide
domain of bone type I collagen. These carboxyterminal telopeptide sequences
are
cross-linked with either lysyl pyridinoline or hydroxylysyl pyridinoline. An
example of such a peptide sequence is represented by the formula:
Asp-Gly-Gln-Hyp-Gly-Ala
Hyp-Glu-Gly-Lys
Gly-Asp-Ala-Gly-Ala-K-Gly-Asp
Glu-K-Ala-His-Asp-Gly-Gly-Arg
I
Glu-K-Ala-His-Asp-Gly-Gly-Arg
where K
I
K
i
K
is hydroxylysyl or lysyl pyridinoline.
, The invention includes a fused cell hydrid which produces monoclonal anti-
bodies specific for the peptide fragment derived from bone collagen
having 3-hydroxypyridinium cross-links. The invention also includes monoclonal
antibodies produced by the fused cell hybrid including those antibodies
coupled to
a detectable marker. Examples of detectable markers include enzymes,
chromophores, fluorophores, coenzymes, enzyme inhibitors, chemiluminescent
materials, paramagnetic metals, spin labels, and radio nucleotides. The
invention
includes a test kit useful for quantitating the amount of peptide fragments
having
_g_
13~~~~9
3-hydroxypyridinium cross-links derived from bone collagen resorption found in
a
body fluid comprising the monoclonal antibody specific for peptide fragments
derived from bone collagen and containing 3-hydroxypyridinium cross-links. The
monoclonal antibodies of this test kit may be coupled to the detectable
markers
described above.
Brief Description of the Drawings
FIGURE 1 is a graph of hydroxypyridinium residues in bone collagen versus
age.
FIGURE 2 is a graph of the ratio of hydroxylysyl pyridinoline (HP) to lysyl
pyridinoline (LP) versus age.
FIGURE 3a is a typical reverse phase HPLC natural fluorescence elution
profile of the aminoterminal telopeptides showing the location of the major
peptide fragment containing 3-hydroxypyridinium cross-links.
FIGURE 3b is a typical reverse phase HPLC natural fluoresence elution
profile of the carboxyterminal telopeptides showing the location of the major
peptide fragment containing 3-hydroxypyridinium cross-links.
FIGURE 4A is a typical reverse phase HPLC elution profile of natural
fluorescence for a hydrolysate of peptide fragments from normal human urine.
FIGURE 4B is a typical reverse phase HPLC elution profile of natural
fluorescence for a hydrolysate of peptide fragments from Paget's disease
patent
urine.
Detailed Description of the Preferred Embodiments
This invention is based on the discovery that both lysyl pyridinoline (LP) and
hydroxylysyl pyridinoline (HP) peptide fragments derived from reabsorbed bone
collagen are excreted in the urine without being metabolized. The invention is
also based on the discovery that no other connective tissues contain
significant
levels of LP and that the ratio of HP to LP in mature bone collagen remains
relatively constant over a person's lifetime.
FIGURE 1 compares the concentration of HP and LP in both cortical and
cancellous human bone with age. It is observed that the concentration of HP
plus
LP cross-links in bone collagen reaches a maximum by age 10 to 15 years and
remains reasonably constant throughout adult life. Furthermore, the ratio of
HP
to LP, shown in FIGURE 2, shows little change throughout life, remaining
constant
at about 3.5 to 1. These baseline data demonstrate that the 3-
hydroxypyridinium
cross-links in bone collagen remain relatively constant and therefore that
body
fluids derived from bone collagen degradation will contain 3-hydroxypyridinium
cross-linked peptide fragments at concentrations proportional to the absolute
rate
of bone resorption.
-9-
,. ,
Since LP is the 3-hydroxypyridinium cross-link unique to bone collagen, the
method for determining the absolute rate of bone resorption, in its simplest
form,
is based on quantitating the concentration of peptide fragments containing
3-hydroxypyridinium cross-links and preferably lysyl pyridinoline (LP) cross-
links
in a body fluid. As used in this description and in the appended claims, by
quanti-
tating is meant measuring by any suitable means, including but not limited to
spectrophotometric, gravimetric, volumetric, coulometric, immunometric,
potentiometric, or amperometric means, the concentration of peptide fragments
containing 3-hydroxypyridinium cross-links in an aliquot of a body fluid.
Suitable
body fluids include urine, serum, and synovial fluid. The preferred body fluid
is
urine.
Since the concentration of urinary peptides will decrease as the volume of
urine increases, it is further preferred that when urine is the body fluid
selected,
the aliquot assayed be from a combined pool of urine collected over a fixed
period
of time, for example, 24 hours. In this way, the absolute rate of bone
resorption is
calculated for a 24 hour period. Alternatively, urinary peptides may be
measured
as a ratio relative to a marker substance found in urine, such as creatinine.
In this
way the urinary index of bone resorption would remain independent of urine
volume.
In one embodiment of the present invention, monoclonal or polyclonal anti-
bodies are produced which are specific to the peptide fragments containing
lysyl
pyridinoline cross-links found in urine. Peptide fragments may be isolated
from
the urine of any patient; however, it is preferred that these peptides are
isolated
from patients with Paget's disease or hyperparathyroidism, due to the high
concentration of peptide fragments found in these patients.
ISOLATION OF URINARY PEPTIDES
Urine from patients with active Paget's disease is dialyzed in reduced
porosity dialysis tubing (>3,500 ~ Spectroporej~at 4°C for 48h to
remove bulk
solutes. Under these conditions the peptides of interest are largely retained.
The
freeze-dried non-diffusate is then eluted (200 mg aliquots) from a column (90
cm x
2.5 cm) of Bio-GeI P2 (200-400 mesh) in 1096 acetic acid at room temperature.
A
region of effluent that combines the cross-linked peptides is defined by
measuring
the fluorescence of collected fractions at 297 nm excitation/395 nm emission,
and
this pool is freeze-dried. Further resolution of this material is obtained on
a
column of Bio-Gel P-4 (200-400 mesh, 90 cm x 2.5 cm) eluted in 1096 acetic
acid.
Two contiguous fraction pools are defined by monitoring the fluorescence of
the
10
eluant above. The earlier fraction is enriched in peptide fragments having two
amino acid sequences that derive from the carboxyterminal telopeptide domain
of
the aI(I) chain of bone type I collagen linked to a third sequence derived
from the
triple-helical body of bone type I collagen. These three peptide sequences are
cross-linked with 3-hydroxypyridinium. The overlapping later fraction is
enriched
in peptide fragments having an amino acid sequence that derives from the
aminoterminal telopeptide domain of bone type I collagen linked through a
3-hydroxypyridinium cross-links. Individual peptides are then resolved from
each
of the two fractions obtained above by ion-exchange HPLC on a TSK DEAE-5-PW
TM
column (Bio Raa~ l.5 cm x 7.5 mm) eluting with a gradient of NaCl (0-0.2M) in
0.02M Tris-HCl, pH 7.5 containing 1096 (v/v) acetonitrile. The amino terminal
telopeptide-based and carboxyterminal telopeptide-based cross-linked peptides
elute in a series of 3-4 peaks of fluorescence between 0.08M and 0.15M NaCl.
The
carboxyterminal telopeptide-based cross-linked peptides elute first as a
series of
fluorescent peaks, and the major and minor aminoterminal telopeptide-based
cross-linked peptides elute toward the end of the gradient as characteristic
peaks. Each of these is collected,.,freeze-dried and chromatographed on a C-18
TM
reverse phase HPLC column (Vydae Z18TP54, 25 cm x 4.6 mm) eluted with a
gradient (0-1096) of acetonitrile: n-propanol (3:1 v/v) in O.O1M
trifluoroacetic
acid. About 100-500 ug of individual peptide fragments containing 3
hydroxypyridinium cross-links can be isolated by this procedure from a single
24h
collection of Paget's urine. Amino acid compositions of the major isolated
peptides confirmed purity and molecular sizes by the whole number
stoichiometry
of recovered amino acids. Aminoterminal sequence analysis by Edman
degradation confirmed the basic core structures suspected from the sequences
of
the known cross-linking sites in type I collagen and from the matching amino
acid
compositions. The aminoterminal telopeptide sequence of the a2(I) chain was
blocked from sequencing analysis due presumably to the known cyclization of
the
aminoterminal glutamine to pyrrolidone carboxylic acid. A typical elution
profile
of aminoterminal telopeptides obtained by the above procedure is shown in
FIGURE 3a. The major peptide fragment obtained has an amino acid composition:
(Asx)2(Glx)2(Gly)5Va1-Tyr-Ser-Thr, where Asx is the amino acid Asp or Asn and
Glx is the amino acid Gln or Glu. The sequence of this peptide is represented
by
Formula III below. Normal urine contains smaller amounts of the peptide
fragment represented by Formula III than the urine of Paget's disease
patients.
r
-11-
FORMULA III
Asp-Glu-K-Ser-Thr-Gly-Gly
Gln-Tyr-Asp-Gly-K-Gly-Val-Gly
K
K
I
where K represents the HP or LP cross-linking amino acids, and
K
Gln represents glutamine or a wholly cyclized pyrrolidone carboxylic acid.
The carboxyterminal telopeptide-based cross-linked peptides resolved by
reverse phase HPLC as described above are shown in FIGURE 3b. As can be seen
from this figure, these peptides are further resolved into a series of
carboxyterminal telopeptides each containing the 3-hydroxypyridinium cross-
links. The major peptide, shown in FIGURE 3b, was analyzed as described above
and was found to have the amino acid composition:
(Asp)5(Glu)4(Gly)1~(His)2(Arg)Z(Hyp)Z(Ala)5. The sequence of this peptide is
represented by formula IV below. It is believed that the other carboxyterminal
telopeptide-based cross-linked peptides appearing as minor peaks in FIGURE 3b
represent additions and deletions of amino acids to the structure shown in
Formula
IV. Any of the peptides contained within these minor peaks are suitable for
use as
immunogens as described below.
FORMULA IV
Asp-Gly-Gln-Hyp-Gly-Ala
I
Hyp-Glu-Gly-Lys
Gly-Asp-Ala-Gly-Ala-K-Gly-Asp
Glu-K-Ala-His-Asp-Gly-Gly-Arg
I
Glu-K-Ala-His-Asp-Gly-Gly-Arg
K
i
where K represents the HP or LP cross-linking amino acids, and
Gln represents glutamine or a wholly cyclized pyrrolidone carboxylic acid.
Equivalents of the peptides represented by the above structures include
those cases where some variation in the urinary peptide structure accrues.
-12- 1~4~'~9~
Examples of variation include amino acid additions to the N and C termini of
Formulae III and IV as well
as some terminal amino acid deletions. Smaller peptide fragments of the
molecule represented by Formula
IV derived from bone readsorption are especially evident in urine. These are
found in the minor peaks of
the carboxytelopeptide fraction seen in Figure 3b and can be identified by
amino acid composition and
sequence analysis. It is anticipatedthat antibodies produced to the haptens
represented by Formulae III
and IV will cross react with urinary peptides of s lightly varied structure.
In some situations it may be
desirable to producepatient-specific antibodies to theurinary peptides derived
from bone resorption. In
these cases the same procedure described above is utilized to isolate urinary
peptides whose structure may
vary slightly from that represented by Formulae III and IV.
IMMUNOLOGICAL PROCEDURE FOR INDEXING BONE RESORPTION
Immunological binding partners capable of specifically binding to peptide
fragments derived from
bone collagen obtained from a physiological fluid can be prepared by methods
well known in the art. The
preferred method for isolating these peptide fragments is described above. By
immunological binding
partners as used herein is meant antibodies and antibody fragments.
Both monoclonal andpolyclonal antibodies specifically binding the peptides
represented by
Formulae III and IV and their equivalents are prepared by methods known in the
art. For example,
LaboratoryTechniques inBiochemistry and MolecularBiology, Campbell, A.M.
(1986) Vol.13 Elsevier.
It is possible to produce antibodies to the above peptides or their
equivalents as isolated.
However, because the molecular weights of these peptide fragments are less
than 5,000, it is preferred
that the hapten be conjugated to a can-ier molecule. Suitable carrier
molecules include, but are not limited
to, bovine serum albumin, ovalbumin, thyroglobulin, and keyhole limpet
hemocyanin (KLH). Preferred
carriers are thyroglobulin and KLH.
It is well known in the art that the orientation of the hapten, as it is bound
to the can ier protein,
is of critical importance to the specificity of the anti-serum. Furthermore,
not all hapten-protein conjugates
are equally successfulimmunogens.
Theselectionofaprotocolforbindingtheparticularhaptentothe
Garner protein therefore depends on the amino acid sequence of the urinary
peptide fragments selected.
For example, if the urinary peptide fragment represented by Formula III is
selected, a preferred protocol
would involve coupling this hapten to keyhole limpet hemocycanin (KLH) , or
other suitable carrier, with
134~'r~~
-13-
carbodiimide. This would ensure that most of the hapten would be conjugated
through the Gly
carboxyterminus, thereby presenting the preferred epitope, namely Tyr and 3-
hydroxypyridinium cross-
link, to the primed vertebrate antibody producing cells (e.g., B-lymphocytes).
Other urinary peptide fragments, depending on the source, may require
different binding protocols.
Accordingly, a number of binding agents may be suitably employed. These
include, but are not limited to,
carbodiimides, glutaraldehyde, mixed anhydrides, as well as both
homobifunctional and heterobifunctional
reagents (see forexample the Pierce 1986-87 catalog, Pierce Chemical Co.,
Rockford, IL). Preferred
binding agents include carbodiimides andheterobifunctional reagents such as m-
Maleimidobenzyl-N-
hydroxysuccinimide ester (MBS).
Methods for binding the hapten to the carrier molecule are known in the art.
See for example
LaboratoryTechniques inBiochemistry and MolecularBiology, Chard, T. (1987)
Vol. 6, Partz Elsevier,
N.Y.
Either monoclonal or polyclonal antibodies to the hapten-Garner molecule
immunogen can be
produced. However, it is preferred that monoclonal antibodies MAb be prepared.
For this reason it
is preferred that immunization be carned out in the mouse. Immunization
protocols for the mouse usually
include an adjuvant. Examples of suitable protocols are described by Chard, T.
(1987) vida supra.
Spleen cells from the immunized mouse are harvested and homogenized and
thereafter fused with cancer
cells in the presence of polyethylene glycol to produce a fused cell hybrid
which produces monoclonal
antibodies specific to peptide fragments derived from bone collagen. Examples
of such peptide fragments
are represented by Formulae III and IV above. Suitable cancer cells include
myeloma, hepatoma,
carcinoma, and sarcoma cells. Detaileddescriptions of this procedure,
including screening protocols,
protocols for growing selected hybrid cells, and harvesting monoclonal
antibodies produced by the
selected hybrid cells are provided in Galfre, G. and Milstein, C. (1981) Meth.
Enzymol. 73, 1. A
preferred preliminary screening protocol involves the use of peptide fragments
derived from bone collagen
resorption and containing 3-hydroxypyridinium cross-links in a solid phase
radioimmunoassay.
Immunological binding partners, especially monoclonal antibodies, produced by
the above
procedures, or equivalent procedures, are employed in various immunometric
assays to quantitate the
concentration of peptide fragments having 3-hydroxypyridinium cross-links
derived from bone collagen
resorption in body fluids. These immunometric assays comprise a monoclonal
antibody or antibody
-14- 1340?~~
fragment coupled to a detectable marker. Examples of suitable detectable
markers include, but are not limited to, enzymes, coenzymes, enzyme
inhibitors,
chromophores, fluorophores, chemiluminescent materials, paramagnetic metals,
spin labels, and radionuclides. Examples of standard immunometric methods
suitable for indexing bone resorption include, but are not limited to, enzyme
linked immunosorbent assay (ELISA) (Ingvall, E. (1981) Meth. Enzymol. 70),
radio-
immunoassay (RIA), and "sandwich" Immuno radiometric assay (IRMA). In its
simplest form, these immunometric methods can be used to determine the
absolute rate of bone resorption by simply contacting a body fluid with the
immunological binding partner specific to a peptide fragment having 3-hydroxy-
pyridinium cross-links derived from bone collagen resorption. It is preferred
that
the immunometric assays described above be conducted directly on untreated
body
fluids. Occasionally, however, contaminating substances may interfere with the
assay, necessitating partial purification of the body fluid. Partial
purification
procedures include, but are not limited to, cartridge adsorption and elution,
mole-
cular sieve chromatography, dialysis, ion exchange, alumina chromatography,
hydroxyapatite chromatography, and combinations thereof.
Test kits, suitable for use in accordance with the present invention, contain
monoclonal antibodies prepared as described above that specifically bind to
pep
tide fragments having 3-hydroxypyridinium cross-links derived from bone
collagen
resorption found in a body fluid. It is preferred that the monoclonal
antibodies of
this test kit be coupled to a detectable marker of the type described above.
ELECTROCHEMICAL PROCEDURE FOR INDEXING BONE RESORPTION
An alternative procedure for indexing bone resorption consists of measuring
a physical property of the peptide fragments having 3-hydroxypyridinium cross-
links. One such physical property suitable for indexing bone resorption relies
upon
electrochemical detection. This method consists of injecting an aliquot of a
body
fluid, such as urine, into an electrochemical detector poised at a redox
potential
suitable for detection of peptides containing the 3-hydroxypyridinium ring.
The 3-hydroxypyridinium ring, being a phenol, is subject to reversible
oxidation,
and therefore the electrochemical detector (e.g., Model 5100A
Couloeherrt''sold by
esa 45 Wiggins Ave., Bedford, MA) is a highly desirable instrument suitable
for
quantitating the concentration of urinary peptides derived from bone
adsorption.
Two basic forms of electrochemical detector are currently commercially
available: amperometric (e.g., BioAnalytical Systems) and coulometric (ESA,
Inc.,
Bedford, MA 01730). Both are suitable for use in accordance with the present
-15 W
invention; however, the latter system is inherently more sensitive and
therefore
preferred since complete oxidation or reduction of the analyzed molecule in
the
column effluent is achieved. In addition, screening or guard electrodes can be
placed "upstream" from the analytical electrode to selectively oxidize or
reduce
interfering substances thereby greatly improving selectivity. Essentially, the
voltage of the analytical electrode is tuned to the redox potential of the
sample
molecule, and one or more pre-treatment cells are set to destroy interferents
in
the sample. In a preferred assay method, a standard current/voltage curve is
established for standard peptides containing lysyl pyridinoline or
hydroxylysyl
pyridinoline in order to determine the proper voltage to set for optimal
sensitivity. This voltage is then modified, depending upon the body fluid, to
minimize interference from contaminants and optimize sensitivity.
Electrochemical detectors, and the optimum conditions for their use are known
to
those skilled in the art. Complex mixtures of body fluids can often be
directly
analyzed with the electrochemical detector without interference. Accordingly,
for most patients no pretreatment of the body fluid is necessary. In some
cases,
however, interfering compounds may reduce the reliability of the measurements.
In such cases, pretreatment of the body fluid (e.g., urine) may be necessary.
Accordingly, in an alternative embodiment of the invention, a body fluid is
first purified prior to electrochemically titrating the purified peptide
fragments.
The purification step may be conducted in a variety of ways, including but not
limited to, dialysis, ion exchange chromatography, alumina chromatography,
hydroxyapatite chromatography, molecular sieve chromatography, or combinations
thereof. In a preferred purification protocol, a measured aliquot (25 ml) of a
24 hour urine sample is dialyzed in reduced porosity dialysis tubing to remove
the
bulk of contaminating fluorescent solutes. The non-diffusate is then
lyophilized,
redissolved in 196 heptafluorobutyric acid (HFBA), an ion pairing solution,
and the
peptides adsorbed on a Waters Sep-Pak~C-18 cartridge. This cartridge is then
washed with 5 ml of 196 HFBA, and then eluted with 3 ml of 5096 methanol in
196
HFBA.
Another preferred method of purification consists of adsorbing a measured
aliquot of urine onto an ion-exchange adsorption filter and eluting the
adsorption
filter with a buffered eluting solution. The eluate fractions containing
peptide
fragments having 3-hydroxypyridinium cross-links are then collected to be
assayed.
Still another preferred method of purification employs molecular sieve
chromatography. For example, an aliquot of urine is applied to a Bio-Gel P2 or
-~Ti~Qe ..rvt~t
1340~1~9
-16-
Sephadex* G-20 column, and the fraction eluting in the 1000-5000 Dalton range
is collected. It will be
obvious to those skilled in the art that a combination of the above methods
may be used to purify or
partially purify urine or other body fluids in order to isolate the peptide
fragments having 3-
hydroxypyridinium cross-links. The purified orpartially purified peptide
fragments obtained by the above
procedures may be subj ected to additional purification procedures, further
processed, or assayed directly
in the partially purified state. Additional purification procedures include
resolving partially purified peptide
fragments employing high performance liquid chromatography (HPLC) or microbore
HPLC when
increased sensitivity is desired. These peptides may then be quantitated by
electrochemical titration. A
preferredelectrochemical titration protocol consists of tuning the redox
potential of the detecting cell of
the electrochemical detector (Coulochem Model 51 OOA) for maximum signal with
pure HP. The detector
is then used to monitor the effluant f mm a C-18 HPLC column used to resolve
the partially purified urinary
peptides.
FLUOROMETRIC PROCEDURE FOR INDEXING BONE RESORPTION
An alternative preferred method for quantitating the concentration of peptide
fragments having 3-
hydroxypyridinium cross-links is to measure the characteristic natural
fluorescence of these peptide
fragments. Forthosebody fluids containing few naturally occurring
fluorescentmaterials otherthan the
3-hydroxypyridiniumcross-links, fluormetric assay may be conducted directly
without further purification
of the body fluid. In this case, peptide fragments are resolved by HPLC and
the natural fluorescence of
the HP and LP amino acid residues is measured at 395 nm upon excitation at 297
nm, essentially as
described by Eyre, D.R., et al., Analyl. Biochem. 137, 380 (1984)
It is preferred, in accordance with the present invention, that the
fluorometric assay be conduced
on urine. Urine, however, usually contains substantial amounts of naturally
occurring fluorescent
contaminants that must be removed prior to conducting the fluorometric assay.
Accordingly, urine samples
are f first partially purified as described above for electrochemical
detection. This partially purified urine
sample can then be fluorometrically assayed as described above. Alternatively,
the HP and LP cross-
linked peptides in the partially purified urine samples or other body fluids
can be hydrolyzed in 6M HCI
at about 108 ° C for approximately 24 hours as described by Eyre, et
al. (1984) vida supra. This process
hydrolyzes the amino acids connected to the lysine precursors of "tripeptide"
HP
* Trade-mark
134~r~9~
-17-
and LP cross-links, producing the free HP and LP amino acids represented by
Formulae I and II. These
small "tripeptides" are then resolved by the techniques decribed above,
preferably by HPLC, and the
natural fluorescence is measured (Ex 297 nm, Em 390 nm).
Optionally, the body fluid (preferably urine) is passed directly through a C-
18 reverse phase
affinity cartridge after adding acetonitrile/methanol 5 to 10% VN. The non-
retentate is adjusted to 0.05-
0.1 OM with a cationic ion-pairing agent such as tetrabutyl ammonium hydroxide
and passed through a
second C-18 reverse phase cartridge. The washedretentate, containing
fluorescentpeptides, from this
second cartridge is eluted with acetonitrile: water (or methanol:water) , and
dried, and the fluorescent
peptides are analyzed by reverse phase HPLC or microbore HPLC using an anionic
ion-pairing agent such
as 0.01 M trif luoroncetic acid in the eluant. Alternatively or included in
this peptide clean-up procedure,
a molecular sieve step employing a Bio-Gel P2 (Bio-Rad labs) or equivalent gel
filtration medium, can be
used.
FIGURE 4A displays the elution profile resolved by reverse phase HPLC of
natural fluorescence
for a hydrolysate of peptide fragments from normal human urine. Measurement of
the integrated area
within the envelope of a given component is used to determine the
concentration of that component within
the sample. The ratio of HP: LP found in normal human urine and urine from
patients having Paget's
disease, FIGURE 4B, are both approximately 4.5:1. This is slightly higher than
the 4:1 ration found in
bone itself (Eyre et al.,1984). The higher ratio found in urine indicates that
a portion of the HP fraction
in urine may come from sources other than bone such as the diet, or other
sources of collagen degradation;
i.e., cartilage catabolism. It is for this reason that it is preferred that LP
which derives only from bone to
be used to provide an absolute index of bone resorption. However, in the
absence of excessive cartilage
degradation such as in rheumatoid arthritis or in cases where bone is rapidly
being adsorbed, HP or a
combination of HP plus LP may be used as an index of bone resorption.
While the invention has been described in conjunction with preferred
embodiments, one of
ordinary skill afterreading the foregoing specification will be able to effect
various changes, substitutions
of equivalents, and alterations to the subj ect matter set forth herein.
Hence, the invention can be practiced
in ways other than those specifically described herein.
X
